Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK.

BACKGROUND: The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy. METHODS: Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas. RESULTS: We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474. CONCLUSIONS: Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.

Orthogonal assays for the identification of inhibitors of the single-stranded nucleic acid binding protein YB-1.

We have previously shown that high expression of the nucleic acid binding factor YB-1 is strongly associated with poor prognosis in a variety of cancer types. The 3-dimensional protein structure of YB-1 has yet to be determined and its role in transcriptional regulation remains elusive. Drug targeting of transcription factors is often thought to be difficult and there are very few published high-throughput screening approaches. YB-1 predominantly binds to single-stranded nucleic acids, adding further difficulty to drug discovery. Therefore, we have developed two novel screening assays to detect compounds that interfere with the transcriptional activation properties of YB-1, both of which may be generalizable to screen for inhibitors of other nucleic acid binding molecules. The first approach is a cell-based luciferase reporter gene assay that measures the level of activation of a fragment of the E2F1 promoter by YB-1. The second approach is a novel application of the AlphaScreen system, to detect interference of YB-1 interaction with a single-stranded DNA binding site. These complementary assays examine YB-1 binding to two discrete nucleic acid sequences using two different luminescent signal outputs and were employed sequentially to screen 7360 small molecule compounds leading to the identification of three putative YB-1 inhibitors.

Synthesis and biological evaluation of solubilized sulfonamide analogues of the phosphatidylinositol 3-kinase inhibitor ZSTK474.

Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation.

Synthesis and Evaluation of Imidazo[1,2-a]pyridine Analogues of the ZSTK474 Class of Phosphatidylinositol 3-Kinase Inhibitors.

Using a scaffold-hopping approach, imidazo[1,2-a]pyridine analogues of the ZSTK474 (benzimidazole) class of phosphatidylinositol 3-kinase (PI3K) inhibitors have been synthesized for biological evaluation. Compounds were prepared using a heteroaryl Heck reaction procedure, involving the palladium-catalysed coupling of 2-(difluoromethyl)imidazo[1,2-a]pyridines with chloro, iodo or trifluoromethanesulfonyloxy (trifloxy) substituted 1,3,5-triazines or pyrimidines, with the iodo intermediates being preferred in terms of higher yields and milder reaction conditions. The new compounds maintain the PI3K isoform selectivity of their benzimidazole analogues, but in general show less potency.

Synthesis and biological evaluation of sulfonamide analogues of the phosphatidylinositol 3-kinase inhibitor ZSTK474.

Replacement of one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 (1) with sulfonamide containing substituents produced a new class of active and potent PI3Kα inhibitors. Solubility issues prevented all but the 6-amino derivative 17 from being evaluated in vivo, but the clear activity of this compound demonstrated that this class of PI3K inhibitor shows great promise.

Combining properties of different classes of PI3Kα inhibitors to understand the molecular features that confer selectivity.

Phosphoinositide 3-kinases (PI3Ks) are major regulators of many cellular functions, and hyperactivation of PI3K cell signalling pathways is a major target for anticancer drug discovery. PI3Kα is the isoform most implicated in cancer, and our aim is to selectively inhibit this isoform, which may be more beneficial than concurrent inhibition of all Class I PI3Ks. We have used structure-guided design to merge high-selectivity and high-affinity characteristics found in existing compounds. Molecular docking, including the prediction of water-mediated interactions, was used to model interactions between the ligands and the PI3Kα affinity pocket. Inhibition was tested using lipid kinase assays, and active compounds were tested for effects on PI3K cell signalling. The first-generation compounds synthesized had IC50 (half maximal inhibitory concentration) values >4 µM for PI3Kα yet were selective for PI3Kα over the other Class I isoforms (ß, δ and γ). The second-generation compounds explored were predicted to better engage the affinity pocket through direct and water-mediated interactions with the enzyme, and the IC50 values decreased by ∼30-fold. Cell signalling analysis showed that some of the new PI3Kα inhibitors were more active in the H1047R mutant bearing cell lines SK-OV-3 and T47D, compared with the E545K mutant harbouring MCF-7 cell line. In conclusion, we have used a structure-based design approach to combine features from two different compound classes to create new PI3Kα-selective inhibitors. This provides new insights into the contribution of different chemical units and interactions with different parts of the active site to the selectivity and potency of PI3Kα inhibitors.

Biological characterization of SN32976, a selective inhibitor of PI3K and mTOR with preferential activity to PI3Kα, in comparison to established pan PI3K inhibitors.

Multiple therapeutic agents have been developed to target the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is frequently dysregulated in cancer promoting tumor growth and survival. These include pan PI3K inhibitors, which target class Ia PI3K isoforms and have largely shown limited single agent activity with narrow therapeutic windows in clinical trials. Here, we characterize SN32976, a novel pan PI3K inhibitor, for its biochemical potency against PI3K isoforms and mTOR, kinase selectivity, cellular activity, pharmacokinetics, pharmacodynamics and antitumor efficacy relative to five clinically-evaluated pan PI3K inhibitors: buparlisib, dactolisib, pictilisib, omipalisib and ZSTK474. SN32976 potently inhibited PI3K isoforms and mTOR, displaying preferential activity for PI3Kα and sparing of PI3Kδ relative to the other inhibitors, while showing less off-target activity than the clinical inhibitors in a panel of 442 kinases. The major metabolites of SN32976 were also potent PI3K inhibitors with similar selectivity for PI3Kα as the parent compound. SN32976 compared favorably with the clinically-evaluated PI3K inhibitors in cellular assays, inhibiting pAKT expression and cell proliferation at nM concentrations, and in animal models, inducing a greater extent and duration of pAKT inhibition in tumors than pictilisib, dactolisib and omipalisib at similarly tolerated dose levels and inhibiting tumor growth to a greater extent than dactolisib and ZSTK474 and with similar efficacy to pictilisib and omipalisib. These results suggest that SN32976 is a promising clinical candidate for cancer therapy with enhanced kinase selectivity and preferential inhibition of PI3Kα compared to first generation pan PI3K inhibitors, while retaining comparable anticancer activity.

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors.

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.

Potentiation of Growth Inhibitory Responses of the mTOR Inhibitor Everolimus by Dual mTORC1/2 Inhibitors in Cultured Breast Cancer Cell Lines.

The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and "triple negative" variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not with AKT or ERK phosphorylation, consistent with the mTOR pathway being a principal target. We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.

Inhibitors of pan-PI3K Signaling Synergize with BRAF or MEK Inhibitors to Prevent BRAF-Mutant Melanoma Cell Growth.

BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms (p110α, A66; p110ß, TGX-221; p110γ, AS-252424; p110δ, idelalisib), or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.

Exploring the isoform selectivity of TGX-221 related pyrido[1,2-a]pyrimidinone-based Class IA PI 3-kinase inhibitors: synthesis, biological evaluation and molecular modelling.

A novel series of TGX-221 analogues was prepared and tested for their potency against the p110α, p110ß, and p110δ isoforms of the PI3K enzyme, and in two cellular assays. The biological results were interpreted in terms of a p110ß comparative model, in order to account for their selectivity towards this isoform. A CH2NH type linker is proposed to allow binding into the specificity pocket proposed to accommodate the high p110ß-selectivity of TGX-221, although there was limited steric tolerance for substituents on the pendant ring with the 2-position most favourable for substitution.

Relationships between signaling pathway usage and sensitivity to a pathway inhibitor: examination of trametinib responses in cultured breast cancer lines.

Cellular signaling pathways involving mTOR, PI3K and ERK have dominated recent studies of breast cancer biology, and inhibitors of these pathways have formed a focus of numerous clinical trials. We have chosen trametinib, a drug targeting MEK in the ERK pathway, to address two questions. Firstly, does inhibition of a signaling pathway, as measured by protein phosphorylation, predict the antiproliferative activity of trametinib? Secondly, do inhibitors of the mTOR and PI3K pathways synergize with trametinib in their effects on cell proliferation? A panel of 30 human breast cancer cell lines was chosen to include lines that could be classified according to whether they were ER and PR positive, HER2 over-expressing, and "triple negative". Everolimus (targeting mTOR), NVP-BEZ235 and GSK2126458 (both targeting PI3K/mTOR) were chosen for combination experiments. Inhibition of cell proliferation was measured by IC50 values and pathway utilization was measured by phosphorylation of signaling kinases. Overall, no correlation was found between trametinib IC50 values and inhibition of ERK signaling. Inhibition of ERK phosphorylation was observed at trametinib concentrations not affecting proliferation, and sensitivity of cell proliferation to trametinib was found in cell lines with low ERK phosphorylation. Evidence was found for synergy between trametinib and either everolimus, NVP-BEZ235 or GSK2126458, but this was cell line specific. The results have implications for the clinical application of PI3K/mTOR and MEK inhibitors.

Extended treatment with selective phosphatidylinositol 3-kinase and mTOR inhibitors has effects on metabolism, growth, behaviour and bone strength.

The class I phosphatidylinositol 3-kinases (PtdIns3Ks) mediate the effects of many hormones and growth factors on a wide range of cellular processes, and activating mutations or gene amplifications of class I PtdIns3K isoforms are known to contribute to oncogenic processes in a range of tumours. Consequently, a number of small-molecule PtdIns3K inhibitors are under development and in clinical trial. The central signalling role of PtdIns3K in many cellular processes suggests there will be on-target side effects associated with the use of these agents. To gain insights into what these might be we investigated the effect of extended daily dosing of eight small-molecule inhibitors of class Ia PtdIns3Ks. Animals were characterized in metabolic cages to analyse food intake, oxygen consumption and movement. Insulin tolerance and body composition were analysed at the end of the experiment, the latter using EchoMRI. Bone volume and strength was assessed by micro-CT and three-point bending, respectively. Surprisingly, after sustained dosing with pan-PtdIns3K inhibitors and selective inhibitors of the p110α isoform there was a resolution of the impairments in insulin tolerance observed in drug-naïve animals treated with the same drugs. However, pan-PtdIns3K inhibitors and selective inhibitors of the p110α have deleterious effects on animal growth, animal behaviour and bone volume and strength. Together, these findings identify a range of on target effects of PtdIns3K inhibitors and suggest use of these drugs in humans may have important adverse effects on metabolism, body composition, behaviour and skeletal health.

Synthesis and biological evaluation of novel phosphatidylinositol 3-kinase inhibitors: Solubilized 4-substituted benzimidazole analogs of 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474).

A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.

IκB kinase ß (IKKß) does not mediate feedback inhibition of the insulin signalling cascade.

In the present study, we have examined whether IKKß [IκB (inhibitor of nuclear factor κB) kinase ß] plays a role in feedback inhibition of the insulin signalling cascade. Insulin induces the phosphorylation of IKKß, in vitro and in vivo, and this effect is dependent on intact signalling via PI3K (phosphoinositide 3-kinase), but not PKB (protein kinase B). To test the hypothesis that insulin activates IKKß as a means of negative feedback, we employed a variety of experimental approaches. First, pharmacological inhibition of IKKß via BMS-345541 did not potentiate insulin-induced IRS1 (insulin receptor substrate 1) tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKß target sites. Secondly, adenovirus-mediated overexpression of wild-type IKKß in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, IRS1 tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signalling was not potentiated in mouse embryonic fibroblasts lacking IKKß. Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKß to IRS1, supporting our findings that IKKß, although activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin signalling cascade.

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors.

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.

Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR.

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.

Identification of cyclohexanone derivatives that act as catalytic inhibitors of topoisomerase I: effects on tamoxifen-resistant MCF-7 cancer cells.

Breast cancer is commonly treated with anti-estrogens or aromatase inhibitors, but resistant disease eventually develops and new therapies for such resistance are of great interest. We have previously isolated several tamoxifen-resistant variant sub-lines of the MCF-7 breast cancer cell line and provided evidence that they arose from expansion of pre-existing minor populations. We have searched for therapeutic agents that exhibit selective growth inhibition of the resistant lines and here investigate 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91). We found that two of the tamoxifen-resistant sub-lines (TamR3 and TamC3) unexpectedly showed increased sensitivity to RL90 and RL91. We utilized growth inhibition assays, flow cytometry and immunoblotting to establish a mechanistic basis for their action. Treated sensitive cells showed S-phase selective DNA damage, as detected by histone H2AX phosphorylation. Cellular responses were similar to those induced by the topoisomerase I poison camptothecin. Although IC(50) values of camptothecin, RL90, RL91 were correlated, studies with purified mammalian topoisomerase I suggested that RL90 and RL91 differed from camptothecin by acting as catalytic topoisomerase I inhibitors. These drugs provide a platform for the further development of DNA damaging drugs that have selective effects on tamoxifen resistant breast cancer cells. The results also raise the question of whether clinical topoisomerase I poisons such as irinotecan and topotecan might be active in the treatment of some types of tamoxifen-resistant cancer.

Effects of acutely inhibiting PI3K isoforms and mTOR on regulation of glucose metabolism in vivo.

In in vitro studies class-I PI3Ks (phosphoinositide 3-kinases), class-II PI3Ks and mTOR (mammalian target of rapamycin) have all been described as having roles in the regulation of glucose metabolism. The relative role each plays in the normal signalling processes regulating glucose metabolism in vivo is less clear. Knockout and knockin mouse models have provided some evidence that the class-I PI3K isoforms p110α, p110ß, and to a lesser extent p110γ, are necessary for processes regulating glucose metabolism and appetite. However, in these models the PI3K activity is chronically reduced. Therefore we analysed the effects of acutely inhibiting PI3K isoforms alone, or PI3K and mTOR, on glucose metabolism and food intake. In the present study impairments in glucose tolerance, insulin tolerance and increased hepatic glucose output were observed in mice treated with the pan-PI3K/mTOR inhibitors PI-103 and NVP-BEZ235. The finding that ZSTK474 has similar effects indicates that these effects are due to inhibition of PI3K rather than mTOR. The p110α-selective inhibitors PIK75 and A66 also induced these phenotypes, but inhibitors of p110ß, p110δ or p110γ induced only minor effects. These drugs caused no significant effects on BMR (basal metabolic rate), O2 consumption or water intake, but BEZ235, PI-103 and PIK75 did cause a small reduction in food consumption. Surprisingly, pan-PI3K inhibitors or p110α inhibitors caused reductions in animal movement, although the cause of this is not clear. Taken together these studies provide pharmacological evidence to support a pre-eminent role for the p110α isoform of PI3K in pathways acutely regulating glucose metabolism.

DMXAA (Vadimezan, ASA404) is a multi-kinase inhibitor targeting VEGFR2 in particular.

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.