Jayne Curry

Fragment-Based Discovery of the Pyrazol-4-Yl Urea (at9283), a Multitargeted Kinase Inhibitor with Potent Aurora Kinase Activity.

Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC(50) approximately 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound 16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.

Aurora B kinase inhibition in mitosis: Strategies for optimizing the use of aurora kinase inhibitors such as AT9283

Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

The heat shock protein 90 inhibitor, AT13387, displays a long duration of action in vitro and in vivo in non‐small cell lung cancer

A ubiquitously expressed chaperone, heat shock protein 90 (HSP90) is of considerable interest as an oncology target because tumor cells and oncogenic proteins are acutely dependent on its activity. AT13387 (2,4‐dihydroxy‐5‐isopropyl‐phenyl)‐[5‐(4‐methyl‐piperazin‐1‐ylmethyl)‐1,3‐dihydro‐isoindol‐2‐yl] methanone, l‐lactic acid salt) a novel, high‐affinity HSP90 inhibitor, which is currently being clinically tested, has shown activity against a wide array of tumor cell lines, including lung cancer cell lines. This inhibitor has induced the degradation of specific HSP90 client proteins for up to 7 days in tumor cell lines in vitro. The primary driver of cell growth (mutant epidermal growth factor receptors) was particularly sensitive to HSP90 inhibition. The long duration of client protein knockdown and suppression of phospho‐signaling seen in vitro after treatment with AT13387 was also apparent in vivo, with client proteins and phospho‐signaling suppressed for up to 72 h in xenograft tumors after treatment with a single dose of AT13387. Pharmacokinetic analyses indicated that while AT13387 was rapidly cleared from blood, its retention in tumor xenografts was markedly extended, and it was efficacious in a range of xenograft models. AT13387s long duration of action enabled, in particular, its efficacious once weekly administration in human lung carcinoma xenografts. The use of longer‐acting HSP90 inhibitors, such as AT13387, on less frequent dosing regimens has the potential to maintain antitumor efficacy as well as minimize systemic exposure and unwanted effects on normal tissues. (Cancer Sci, 2012; 103: 522–527)

The HSP90 Inhibitor, AT13387, Is Effective against Imatinib-Sensitive and -Resistant Gastrointestinal Stromal Tumor Models

The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib. Mol Cancer Ther; 11(8); 1799–808. ©2012 AACR.

AT9283, a potent inhibitor of the Aurora kinases and Jak2, has therapeutic potential in myeloproliferative disorders.

Constitutive activation of Janus kinase (Jak) 2 is the most prevalent pathogenic event observed in the myeloproliferative disorders (MPD), suggesting that inhibitors of Jak2 may prove valuable in their management. Inhibition of the Aurora kinases has also proven to be an effective therapeutic strategy in a number of haematological malignancies. AT9283 is a multi‐targeted kinase inhibitor with potent activity against Jak2 and Aurora kinases A and B, and is currently being evaluated in clinical trials. To investigate the therapeutic potential of AT9283 in the MPD we studied its activity in a number of Jak2‐dependent systems. AT9283 potently inhibited proliferation and Jak2‐related signalling in Jak2‐dependent cell lines as well as inhibiting the formation of erythroid colonies from haematopoietic progenitors isolated from MPD patients with Jak2 mutations. The compound also demonstrated significant therapeutic potential in vivo in an ETV6‐JAK2 (TEL‐JAK2) murine leukaemia model. Inhibition of both Jak2 and Aurora B was observed in the model systems used, indicating a dual mechanism of action. Our results suggest that AT9283 may be a valuable therapy in patients with MPD and that the dual inhibition of Jak2 and the Aurora kinases may potentially offer combinatorial efficacy in the treatment of these diseases.

A novel Hsp90 inhibitor AT13387 induces senescence in EBV-positive nasopharyngeal carcinoma cells and suppresses tumor formation

BackgroundNasopharyngeal carcinoma (NPC) is an epithelial malignancy strongly associated with Epstein-Barr virus (EBV). AT13387 is a novel heat shock protein 90 (Hsp90) inhibitor, which inhibits the chaperone function of Hsp90 and reduces expression of Hsp90-dependent client oncoproteins. This study aimed to evaluate both the in vitro and in vivo antitumor effects of AT13387 in the EBV-positive NPC cell line C666-1.ResultsOur results showed that AT13387 inhibited C666-1 cell growth and induced cellular senescence with the downregulation of multiple Hsp90 client oncoproteins EGFR, AKT, CDK4, and restored the protein expression of negative cell cycle regulator p27. We also studied the ability of AT13387 to restore p27 expression by downregulation of AKT and the p27 ubiquitin mediator, Skp2, using AKT inhibitor and Skp2 siRNA. In the functional study, AT13387 inhibited cell migration with downregulation of a cell migration regulator, HDAC6, and increased the acetylation and stabilization of α-tubulin. We also examined the effect of AT13387 on putative cancer stem cells (CSC) by 3-D tumor sphere formation assay. AT13387 effectively reduced both the number and size of C666-1 tumor spheres with decreased expression of NPC CSC-like markers CD44 and SOX2. In the in vivo study, AT13387 significantly suppressed tumor formation in C666-1 NPC xenografts.ConclusionAT13387 suppressed cell growth, cell migration, tumor sphere formation and induced cellular senescence on EBV-positive NPC cell line C666-1. Also, the antitumor effect of AT13387 was demonstrated in an in vivo model. This study provided experimental evidence for the preclinical value of using AT13387 as an effective antitumor agent in treatment of NPC.

Abstract 2772: AT13387, an HSP90 inhibitor, is effective in both vemurafenib-sensitive and -resistant melanoma models

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Mutations in BRAF are found in approximately 50% of melanomas. Vemurafenib, the selective mutant BRAF inhibitor, is an effective treatment in this disease but is limited by the onset of resistance. A number of mechanisms of resistance to RAF inhibitors have been described, including alternative mechanisms for activating the MEK-ERK and AKT pathways. HSP90 inhibitors affect clients in both these pathways and so it has been suggested that HSP90 inhibition is a potential mechanism for overcoming resistance to RAF inhibitors in melanoma. Results: AT13387 is a fragment-derived, potent HSP90 inhibitor, which is currently being evaluated in clinical trials. AT13387 inhibited cell proliferation in a range of melanoma cell lines, including cell lines with different mechanisms of resistance to vemurafenib (See Table). AT13387 treatment of melanoma cell lines resulted in the induction of HSP70, a marker of HSP90 inhibition, and depletion of HSP90 client proteins including BRAF and AKT. The levels of phospho-ERK, phospho-AKT and phospho-S6 were also depleted in both vemurafenib-sensitive (A375) and vemurafenib-resistant (A2058) lines indicating that the MEK-ERK and AKT signaling pathways were both inhibited. This demonstrates that vemurafenib resistance mediated by upregulation of the AKT pathway can be overcome by HSP90 inhibition. In vivo, AT13387 significantly inhibited growth of an A375 melanoma xenograft. Client proteins, including BRAF, CRAF and AKT were depleted in xenograft tissue and the MEK-ERK and AKT signaling pathways were again inhibited. Conclusions: These results demonstrate the activity of AT13387 in both vemurafenib-sensitive and -resistant models and show that HSP90 inhibition results in downregulation of signaling pathways that may be activated as a result of resistance to RAF inhibitors. These data support further clinical evaluation of AT13387 in melanoma. ![Figure][1] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2772. doi:1538-7445.AM2012-2772 [1]: pending:yes

Abstract A217: Comparison of long‐term pharmacodynamic actions of the synthetic small molecule HSP90 inhibitor AT13387 in multiple xenograft models

AT13387 is a novel small molecule inhibitor of HSP90. a member of a family of molecular chaperones. Previously we highlighted an association between the high affinity binding of AT13387 to the N‐terminal ATPase domain of HSP90 and the duration of target inhibition in tumor cell lines in vitro. Further, AT13387 was shown to inhibit HSP90 and deplete client proteins in tumor xenografts longer than other, lower affinity inhibitors in the class. Here we have expanded the investigation to a wider number of tumor cell lines and to in vivo xenograft models and demonstrate that AT13387 has an extended pharmacodynamic action in tumors compared to other HSP90 inhibitors. We reason that the cumulative effects of these properties allow for less frequent dosing thus maximising efficacy whilst minimising systemic exposure and the potential for side effects. This study reports extended inhibition of HSP90 by AT13387 in a wider range of tumor cell lines in vitro. A 24hr exposure of A375 (melanoma) cells to AT13387 suppressed the expression of client proteins for 72 hrs or more. However in other cell lines such as NCI‐H1975 (lung) and BT474 (breast), the suppression of client proteins by AT13387 was found to last in excess of 7 days. The pharmacodynamic action of AT13387 in vivo has been compared with that of 17‐AAG and SNX‐5422 in A375 and NCI‐H1975 xenografts in nude mice. Following a single dose of each agent, we have investigated and compared the time course of the suppression of levels of several client proteins (e.g. AKT, CDK4) and the phosphorylation of key growth/survival signalling components (e.g. pERK, pS6, pAKT). These effects were rapidly induced in tumors following treatment with AT13387 and levels remained suppressed for up to 96 hrs. The durability of the AT13387 effects was significantly greater than for the other competitor compounds. Investigation of tumor growth in these models demonstrated that the longer pharmacodynamic action of AT13387 ensured that efficacy could be maintained on a once weekly schedule, whereas such a schedule for the other agents resulted in a significant loss of their anti‐tumor effects. These data provide further support for the potential benefit of long acting HSP90 inhibitors as a way of maintaining anti‐tumor effects whilst minimising potential for undesirable effects associated with systemic exposure. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A217.

Abstract 2315: Pharmacodynamic and antitumor activity of fragment-derived inhibitors of MetAP2 in tumor xenografts

Background: Methionine aminopeptidase (MetAP) 2 is one of the two eukaryotic enzymes responsible for cleaving the N-terminal methionine from newly synthesized polypeptides to allow further post-translational modifications such as myristoylation to take place. The enzyme is the target of the anti-angiogenic natural product, fumagillin and so is believed to play a role in angiogenesis. Fumagillin analogues have shown activity in several disease models including oncology, inflammation and obesity indicating MetAP2 is a promising target in a number of indications. The semi-synthetic fumagillin analogue, TNP470, has shown activity in a Phase I/II cancer trial, suggesting that MetAP2 is a good oncology target. Here we describe the discovery, optimization and anti-tumor activity of fragment-derived MetAP2 inhibitors. Results: We identified fragment hits to MetAP2 using our fragment-based screening approach, Pyramid™. These were optimized by structure-based drug design to novel, potent lead compounds with sub-100 nM potency against the isolated MetAP2 enzyme. The two most advanced compounds inhibited proliferation of HUVECs with potencies of 130 nM and 300 nM. Levels of the MetAP2 substrate, Met-14-3-3, were shown to increase on treatment of HUVECs with these compounds indicating that Met-14-3-3 was not being processed and hence MetAP2 was being inhibited. The compounds also inhibited HUVEC tubule formation demonstrating their anti-angiogenic properties. The two lead compounds were further tested in vivo. Both compounds were well tolerated at doses up to 200 mg/kg bid. Levels of Met-14-3-3 were seen to increase in the thymus and spleen (where high expression of MetAP2 has been reported) of mice treated with the compounds, indicating again that MetAP2 was being inhibited in these tissues. Compound 1 was tested in a mouse HCT116 xenograft model. Mice were subcutaneously inoculated with HCT116 cells and oral dosing at 200 mg/kg bid started one day later. Tumor growth was inhibited in treated mice compared with control and growth inhibition was greater in Compound 1 treated mice (T/C 46%) compared with mice treated with 30 mg/kg TNP470 subcutaneously q2d (T/C 61%). Conclusions: The compounds described here are novel small-molecule inhibitors of MetAP2. Their promising tumor growth inhibitory properties merit their testing in further tumor models and potentially other indications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2315. doi:1538-7445.AM2012-2315

Abstract A217: The HSP90 inhibitor AT13387 demonstrates potent antitumor activity in both imatinib-sensitive and imatinib-resistant gastrointestinal stromal tumor models.

Background: Activating mutations in the receptor tyrosine kinase, KIT, are found in the majority of gastrointestinal stromal tumors (GIST) and further secondary resistance mutations in KIT frequently arise upon treatment with tyrosine kinase inhibitors such as imatinib. KIT and its mutant forms are sensitive clients of HSP90 and it has been suggested that HSP90 inhibition could be an effective treatment for both imatinib-sensitive and imatinib-resistant GIST, which can occur simultaneously in different clones within the same patient. Methods: AT13387 is a fragment-derived, potent HSP90 inhibitor, which is currently being evaluated in clinical trials. To evaluate its anti-tumor activity against GIST, AT13387 was tested in both imatinib-sensitive (GIST882, GIST-PSW) and imatinib-resistant (GIST430, GIST48) in vitro and in vivo GIST models. Results: AT13387 inhibited the proliferation of GIST cell lines at sub-100 nM potencies, irrespective of their sensitivity to imatinib. Treatment of GIST882, GIST430 and GIST48 cells with AT13387 induced HSP70, a marker of HSP90 inhibition, whilst reducing the levels of the client proteins KIT, AKT and their phosphorylated forms. A concomitant decrease in the levels of phospho-ERK and phospho-S6 demonstrated that KIT signaling was being inhibited in all cell lines, whilst an increase in cleaved PARP indicated apoptosis. In contrast, treatment with imatinib inhibited KIT signaling only in the imatinib-sensitive line, GIST882. In vivo, the efficacy of AT13387 was tested in imatinib-sensitive (GIST-PSW) and imatinib-resistant (GIST430) xenograft models. AT13387, dosed once a week, inhibited the growth of both xenografts; depletion of phospho-KIT and inhibition of KIT signaling were again seen in these tumors. As expected, treatment with imatinib caused significant regression of the GIST-PSW tumors but not of GIST430. The combination of imatinib and AT13387 significantly enhanced tumor growth inhibition (T/C 21%) over either of the monotherapies (T/C 30% for AT13387, 46% for imatinib) in the GIST430 xenograft. Importantly, the combination was well tolerated. Conclusions: AT13387 is currently being evaluated in a Phase II GIST trial both as single agent and in combination with imatinib. These results support the potential efficacy of the compound in both imatinib-sensitive and imatinib-resistant GIST and its current testing in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A217.