Shuichi Ohkubo

TAS-116, a Highly Selective Inhibitor of Heat Shock Protein 90α and β, Demonstrates Potent Antitumor Activity and Minimal Ocular Toxicity in Preclinical Models

The molecular chaperone HSP90 plays a crucial role in cancer cell growth and survival by stabilizing cancer-related proteins. A number of HSP90 inhibitors have been developed clinically for cancer therapy; however, potential off-target and/or HSP90-related toxicities have proved problematic. The 4-(1H-pyrazolo[3,4-b]pyridine-1-yl)benzamide TAS-116 is a selective inhibitor of cytosolic HSP90α and β that does not inhibit HSP90 paralogs such as endoplasmic reticulum GRP94 or mitochondrial TRAP1. Oral administration of TAS-116 led to tumor shrinkage in human tumor xenograft mouse models accompanied by depletion of multiple HSP90 clients, demonstrating that the inhibition of HSP90α and β alone was sufficient to exert antitumor activity in certain tumor models. One of the most notable HSP90-related adverse events universally observed to differing degrees in the clinical setting is visual disturbance. A two-week administration of the isoxazole resorcinol NVP-AUY922, an HSP90 inhibitor, caused marked degeneration and disarrangement of the outer nuclear layer of the retina and induced photoreceptor cell death in rats. In contrast, TAS-116 did not produce detectable photoreceptor injury in rats, probably due to its lower distribution in retinal tissue. Importantly, in a rat model, the antitumor activity of TAS-116 was accompanied by a higher distribution of the compound in subcutaneously xenografted NCI-H1975 non–small cell lung carcinoma tumors than in retina. Moreover, TAS-116 showed activity against orthotopically transplanted NCI-H1975 lung tumors. Together, these data suggest that TAS-116 has a potential to maximize antitumor activity while minimizing adverse effects such as visual disturbances that are observed with other compounds of this class. Mol Cancer Ther; 14(1); 14–22. ©2014 AACR.

A novel cinnamic acid derivative that inhibits Cdc25 dual-specificity phosphatase activity

The Cdc25 dual‐specificity phosphatases are key regulators of cell cycle progression through activation of cyclin‐dependent kinases (Cdk). Three homologs exist in humans: Cdc25A, Cdc25B, and Cdc25C. Cdc25A and Cdc25B have oncogenic properties and are overexpressed in some types of tumors. Compounds that inhibit Cdc25 dual‐specificity phosphatase activity might thus be potent anticancer agents. We screened several hundred compounds in a library using an in vitro phosphatase assay, with colorimetric measurement of the conversion of p‐nitrophenyl phosphate (pNPP) to p‐nitrophenol by the catalytic domain of recombinant human Cdc25, and discovered TPY‐835, which inhibits Cdc25A and Cdc25B activity (IC50 = 5.1 and 5.7 µM, respectively). TPY‐835 had mixed inhibition kinetics for Cdc25A and Cdc25B. TPY‐835 caused cell cycle arrest in the G1 phase in human lung cancer cells (A549 and SBC‐5) but not cell cycle arrest in the G2/M phase. After treatment with TPY‐835, the activation of Cdk2 was suppressed and phosphorylation of the retinoblastoma (Rb) protein was decreased in SBC‐5 cells. In addition, TPY‐835 induced an increase of the sub‐G1 phase cell population after 48–72 h treatment. The growth inhibitory effects of TPY‐835 against cisplatin (CDDP)‐, camptothecin‐ and 5‐FU‐resistant cell lines are comparable to the growth inhibitory effect on their parental lines, thus indicating that TPY‐835 did not show cross‐resistance to these cell lines. These results suggest that TPY‐835 is a promising candidate for constructing a novel class of antitumor agents that can control the cell cycle progression of cancer cells. (Cancer Sci 2005; 96: 614–619)

Substrate Phage as a Tool to Identify Novel Substrate Sequences of Proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.

Abstract B87: First-in-human phase I dose escalation study of TAS-116, a novel, orally active HSP90α and HSP90β selective inhibitor, in patients with advanced solid tumors

Purpose: TAS-116 is a potent non-ansamycin, non-purine, and non-resorcinol orally available highly selective inhibitor of HSP90α and HSP90β. The primary objective of this phase I first-in-human study was to determine maximum-tolerated dose (MTD) of two TAS-116 dosing regimens and estimate recommended phase 2 dose (RP2D) in patients with advanced solid tumors. Secondary objective was to investigate safety, pharmacokinetic and pharmacodynamic profiles. Methods: Eligible patients included those 20 years or older of age with advanced solid tumor that was refractory to conventional treatment or for which no standard therapy existed and ECOG performance status of 0 or 1. TAS-116 was administered orally once daily (Step1) and then subsequently every other day (Step 2) of a 21-day cycle in an accelerated titration dose escalation and a standard 3+3 dose escalation, respectively. Pharmacokinetics was measured after single and multiple dose. Pharmacodymanics was assessed by HSP70 induction in peripheral blood mononuclear cells (PBMCs). Results: As of 17 Jun 2015, 16 patients were enrolled in Step 1 followed by 3 in Step 2. (57% male, median age of 56, 70% PS 0). The numbers of types of tumors were 13 with non-small cell lung cancer (NSCLC), 2 with gastrointestinal stromal tumor (GIST), 2 with thymic carcinoma, and 2 with others. In Step 1, TAS-116 was evaluated at 7 dose levels of 4.8 to 150.5 mg/m2. The most common treatment related adverse events were increased creatinine, diarrhea, increased alkaline phosphatase, night blindness, nausea, anorexia and skin rash. Dose limiting toxicities (DLTs) occurred in 4 patients at 107.5 mg/m2 and 150.5 mg/m2, with grade 3 night blindness and visual disorder, grade 3 AST/ ALT/ gamma-GTP elevations, and grade 3 anorexia. Night blindness and visual disorder including grade 3 were reversible when study drug was interrupted. 107.5 mg/m2 was determined as the once daily MTD and additional patients are being investigated at this dose to confirm tolerability. TAS-116 exposure was increased dose-dependently without unexpected accumulation after repeated administrations. HSP70 induction in PBMC was observed, indicative of target engagement. Of 16 evaluable patients on once daily dosing, there was two confirmed partial response (GIST and NSCLC). Five patients had stable disease more than 12 weeks. Conclusions: TAS-116 administered once daily has an acceptable toxicity profile and the MTD was determined as 107.5 mg/m2. Preliminary antitumor activity was demonstrated with evidence of target engagement. The dose escalation study administered every other day is ongoing and the MTD will be determined. Clinical trial information: JapicCTI-142444. Citation Format: Akihiko Shimomura, Atsushi Horiike, Yuichi Tambo, Fumiyoshi Ohyanagi, Noriko Yanagitani, Satoru Kitazono, Yutaka Fujiwara, Yuko Tanabe, Shuichi Ohkubo, Noboru Yamamoto, Makoto Nishio. First-in-human phase I dose escalation study of TAS-116, a novel, orally active HSP90α and HSP90β selective inhibitor, in patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B87.

Abstract C177: TAS4464, a novel and highly potent inhibitor of NEDD8 activating enzyme, overcomes insensitivity to BTK, PI3Kδ, and IRAK4 inhibitors in activated B-cell like diffuse large B-cell lymphoma via inactivation of the NF-κB pathway

BACKGROUND: NEDD8 activating enzyme (NAE) regulates NEDD8 conjugation in neddylation pathway, a part of the ubiquitin-proteasome system, and controls cell cycle progression, DNA replication, and signal transduction via activation of cullin-RING ligases (CRLs) and subsequent degradation of their substrates. Through one such pathway, the constitutively active nuclear factor κB (NF-κB) triggers the progression of some types of hematological cancer. Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) remains the least curable form of DLBCL despite recent therapeutic advances. Because either phosphatidylinositol-3 kinase (PI3K)/AKT signaling or NF-κB signaling constitutively activated by B cell receptor signaling and MYD88 are reported to promote cancer cell growth and survival in ABC-DLBCL, the key kinases in these pathways [PI3Kδ, Bruton9s tyrosine kinase (BTK), and Interleukin-1 receptor-associated kinases (IRAK)] are considered to be potential targets for ABC-DLBCL therapy. We previously reported an NAE inhibitor, TAS4464, that could inactivate NF-κB by accumulating phosphorylated IκBα (p-IκBα). Here, we report on the possible application of TAS4464 to treat ABC-DLBCL that is insensitive to kinase inhibitors. MATERIALS AND METHODS: Human ABC-DLBCL cell lines, OCI-LY10, SU-DHL-2, and TMD8 were used to evaluate the biological activity of TAS4464. Intracellular ATP levels were measured to assess in vitro cell growth. The effects of TAS4464 on NEDD8 conjugation and levels of CRLs substrates were evaluated by Western blot analysis. The levels of NF-κB-targeted gene transcripts were assessed by qRT-PCR. The antitumor activities of TAS4464 were evaluated in both subcutaneous and systemic xenograft model of TMD8. RESULTS: TAS4464 led to growth arrest and death in ABC-DLBCL cell lines in the nanomolar range through inhibition of cullin neddylation. Similar effects were also observed in a cell line insensitive to the kinase inhibitors that inhibit divergent activator of NF-κB. qRT-PCR analysis revealed TAS4464 inhibited the NF-κB pathway in all ABC-DLBCL cell lines tested through accumulation of p-IκBα, which directly inhibit NF-κB activity. Intravenous administration of TAS4464 showed strong antitumor activity in a TMD8 xenograft model, with inhibited cullin neddylation and activation of caspases 3 and 8 in the tumors. Futhermore, in a TMD8 systemic model, intravenous administration of TAS4464 prolonged mice survival more than two-fold. CONCLUSION: TAS4464 leads to cell growth through inhibition of NF-κB pathway regardless of the activation signaling pathway of NF-κB pathway. Though NF-kB activation signaling is complicated, the downstream inhibition of NF-κB pathway by TAS4464 indicated the potential for treatment of ABC-DLBCL in which NF-kB was dominant for their survival. In addition, TAS4464 demonstrates marked antitumor activity and survival benefit in a human ABC-DLBCL model. Therefore, TAS4464 may be a valuable addition to current options for chemotherapy in ABC-DLBCL. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Shingo Tsuji, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel and highly potent inhibitor of NEDD8 activating enzyme, overcomes insensitivity to BTK, PI3Kδ, and IRAK4 inhibitors in activated B-cell like diffuse large B-cell lymphoma via inactivation of the NF-κB pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C177.

Abstract C176: TAS4464, a novel, highly potent, and selective inhibitor of NEDD8 activating enzyme demonstrates sustained target inhibition and antitumor activity in a preclinical model

BACKGROUND: NEDD8 activating enzyme (NAE) is an essential E1 enzyme in the NEDD8 conjugation (neddylation) pathway, which controls cancer cell growth and survival through activation of cullin-RING ligase complexes (CRLs). Therefore, NAE is considered to be a promising target for cancer therapy. Here we describe the profile of a novel, highly potent, and selective NAE inhibitor, TAS4464, which inactivates CRLs in tumors and has strong antitumor efficacy in a preclinical model. MATERIALS AND METHODS: The selectivity of TAS4464 to E1 enzymes were assessed by various ubiquitin-like protein conjugation assays. The effects of TAS4464 on NEDD8 conjugation and the amounts of CRL substrates were evaluated by Western blot analysis. Cell viability was measured with a Cell Titer-Glo assay. The antitumor activity of TAS4464 was evaluated in an acute lymphoblastic leukemia (CCRF-CEM) xenograft model. RESULTS: We demonstrated that TAS4464 was a mechanism-based NAE inhibitor. TAS4464 formed an NEDD8-TAS4464 adduct that could achieve nanomolar inhibition of NAE. TAS4464 selectively inhibited NAE enzyme activity relative to the other E1s, ubiquitin activating enzyme (UAE) and SUMO activating enzyme (SAE). TAS4464 treatment inhibited cullin neddylation and induced accumulation of key CRL substrate proteins such as CDT1, p27, and phosphorylated IκB in human cancer cell lines. Enzyme inhibitory and cellular NAE inhibitory activities of TAS4464 were higher than those of a known NAE inhibitor, MLN4924. Cytotoxicity profiling of TAS4464 revealed widespread antiproliferative activity against a panel of cancer cell lines. Almost all cell lines derived from hematological malignancies were highly sensitive to TAS4464. Whereas carbonic anhydrase inhibition by MLN4924 resulted in accumulation in red blood cells, the blood-to-plasma ratio of TAS4464 was close to equivalent because of higher selectivity. In addition, TAS4464 did not inhibit major cytochrome p450 enzymes and showed good metabolic stability in isolated hepatocytes in vitro . The pharmacodynamic action of TAS4464 in tumors assessed by measuring the amount of cullin-NEDD8 conjugation in CCRF-CEM xenografts revealed strong cullin neddylation inhibitory activity of TAS4464; the inhibitory activity was sustained longer than that of MLN4924. In the CCRF-CEM xenograft model, weekly administration of TAS4464 at a dose of 100 mg/kg was more efficacious than twice-weekly administration of MLN4924 at a dose of 120 mg/kg: TAS4464 led to complete tumor regression without marked weight loss. CONCLUSION: TAS4464 is the most potent and selective NAE inhibitor reported to date, with good pharmaceutical properties and superior efficacy. Hematological cancer cell lines are highly sensitive to TAS4464. Based on its promising preclinical activity, a phase 1 clinical trial of TAS4464 is being planned. Citation Format: Chihoko Yoshimura, Hiromi Muraoka, Hiroaki Ochiiwa, Shingo Tsuji, Akihiro Hashimoto, Toru Takenaka, Hiromi Kazuno, Keiji Ishida, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel, highly potent, and selective inhibitor of NEDD8 activating enzyme demonstrates sustained target inhibition and antitumor activity in a preclinical model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C176.

Abstract C178: TAS4464, a novel NEDD8 activating enzyme inhibitor, potently induces cell death via both intrinsic and extrinsic apoptotic pathways in acute myeloid leukemia

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by numerous genetic mutations and chromosomal abnormalities. Current chemotherapies have shown limited efficacy in AML patients, most of whom develop chemoresistance and relapse repeatedly. Because the expression of anti-apoptotic proteins reportedly correlates with poor prognosis in AML, the modulation of apoptosis-related proteins emerges as a promising treatment strategy. Apoptosis occurs mainly via two signaling pathways. In the mitochondria-mediated intrinsic apoptotic pathway, the release of cytochrome c triggers caspase-9 activation, which subsequently activates effector caspases (e.g., caspase-3). In the death receptor-mediated extrinsic apoptotic pathway, the binding of death receptor ligands to their receptors activates caspase-8 and, in turn, effector caspases, as in the intrinsic pathway. NEDD8 activating enzyme (NAE) inhibitors, which act on the NEDD8 conjugation pathway, induce quantitative changes in the substrates of E3 cullin-RING ligases (CRLs) and cause apoptosis in AML. Here, we describe the mechanism by which our highly potent and selective NAE inhibitor TAS4464 promotes apoptosis in AML. MATERIALS AND METHODS: Intracellular ATP levels were measured to assess in vitro cell growth. The effects of TAS4464 on NEDD8 conjugation, the protein levels of CRL substrates, and the activation of various caspases were evaluated by means of Western blotting. The transcription levels of the Noxa and c-FLIP genes were assessed by using qRT-PCR. The antitumor activity and in vivo pharmacodynamic activity of TAS4464 were evaluated in a THP-1 xenograft model. RESULTS: TAS4464 led to cell growth arrest and cell death in AML cell lines with various genetic backgrounds, including the MLL-AF9 fusion gene and FLT3-ITD mutation. TAS4464 activated both caspase-9 (intrinsic apoptotic pathway) and caspase-8 (extrinsic apoptotic pathway); combined treatment with inhibitors of these caspases markedly diminished the TAS4464-associated induction of apoptosis. By analyzing apoptosis-related factors in each apoptotic pathway, we found that TAS4464 increased the pro-apoptotic factor Noxa protein (intrinsic apoptotic pathway) and decreased the anti-apoptotic factor c-FLIP protein (extrinsic apoptotic pathway) with changes evident at the mRNA transcriptional level. In addition, treatment with TAS4464 led to accumulation of the CRL substrate c-Myc, and c-Myc knockdown by siRNA ablated the TAS4464-induced decrease in cFLIP protein. Intravenous administration of TAS4464 on day 1, 3 and 5 for 3 weeks resulted in tumor growth inhibition (TGI) of 100% including complete response in human THP-1 xenograft model, compared with twice-weekly administration of cytarabine that resulted in a TGI of only 6%. Pharmacodynamics analysis revealed that TAS4464 inhibited cullin neddylation and induced activation of caspases in THP-1 xenografted tumor. CONCLUSION: TAS4464 exerts a strong apoptosis-inducing effect in AML cell lines via both intrinsic and extrinsic apoptotic pathway by modulating apoptosis-related proteins. In addition, TAS4464 demonstrates marked antitumor activity in the THP-1 xenograft model. Given its potent preclinical activities, TAS4464 is a promising agent for treating AML. Citation Format: Hiroaki Ochiiwa, Chihoko Yoshimura, Hiromi Muraoka, Keiji Ishida, Tomonori Haruma, Shingo Tsuji, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel NEDD8 activating enzyme inhibitor, potently induces cell death via both intrinsic and extrinsic apoptotic pathways in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C178.

Abstract 1730: TAS4464, a novel highly potent NEDD8 activating enzyme inhibitor, demonstrates antitumor activity in multiple myeloma through the inactivation of NF-κB pathways

Background: NEDD8 activating enzyme E1 (NAE) regulates NEDD8 conjugation in the ubiquitin-proteasome system. NAE activates cullin-RING ligases (CRL) and controls the degradation of their substrates, which play key roles in cell cycle progression, DNA replication and signal transduction via various pathways. Through one such pathway, the constitutively active nuclear factor κB (NF-κB) triggers the progression of multiple myeloma (MM). MM is highly treatable but rarely curable, and patients with refractory MM would benefit from additional therapeutic options. NF-κB is activated through two signaling cascades, canonical and non-canonical pathways, which are regulated by the CRL substrates, phosphorylated IκBα (p-IκBα) and phosphorylated p100 (p-p100), respectively. Here, we report on the possible use of an NAE inhibitor that we discovered, TAS4464, to treat MM. Materials and methods: TAS4464 was designed and synthesized at Taiho Pharmaceutical Co., Ltd. Human MM cell lines, KMS-11, KMS-12-BM, KMS-26, KMS-34, and MM.1S were used to evaluate the biological activities of TAS4464. Intracellular ATP levels were measured to assess in vitro cell growth. Effects on the cell cycle were analyzed by using fluorescence-activated cell sorting. The effects of TAS4464 on NEDD8 conjugation and the levels of CRL substrates were evaluated by means of Western blotting. The transcription levels of NF-κB-targeted genes were assessed by using qRT-PCR. Activated p65 and RelB (subunits of NF-κB) were quantified by using commercial DNA-binding ELISA kits. The antitumor activity of TAS4464 was evaluated in an MM.1S xenograft model. Results: TAS4464 inhibited cullin neddylation, leading to cell-growth arrest and apoptosis in MM cell lines in the nanomolar range. These effects were not attenuated after stimulation with interleukin 6 or insulin-like growth factor 1 or in co-culture with stroma cells. TAS4464 led to the accumulation of p-IκBα and p-p100 with subsequent suppression both p65 and RelB activity, whereas bortezomib suppressed only RelB activity. Once-weekly intravenous administration of TAS4464 showed much stronger antitumor activity than that of bortezomib in an MM.1S xenograft model, with inhibited cullin neddylation and induced the cleaved forms of caspases 3 and 8 in the tumors.Conclusion: TAS4464 demonstrated marked antitumor activity in a human MM xenograft model. In addition, TAS4464 inhibited canonical as well as non-canonical pathways of NF-κB, whereas bortezomib inhibited only non-canonical pathway of NF-κB. Therefore, TAS4464 may be a valuable addition to current options for the chemotherapy of MM. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Shingo Tsuji, Takamasa Suzuki, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Yoshikazu Iwasawa, Teruhiro Utsugi. TAS4464, a novel highly potent NEDD8 activating enzyme inhibitor, demonstrates antitumor activity in multiple myeloma through the inactivation of NF-κB pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1730. doi:10.1158/1538-7445.AM2015-1730

Abstract C127: TAS-116, an orally available HSP90α/β selective inhibitor: Synthesis and biological evaluation.

Background: The molecular chaperone HSP90 has long been considered a promising target for cancer therapy; however, several HSP90 inhibitors have demonstrated excessive adverse effects in clinical trials potentially involving off-target and/or HSP90-related toxicities. TAS-116 is a novel, potent and orally active HSP90α/β inhibitor discovered by chemical optimization of an initial hit compound having a 4-(1H-indol-1-yl)benzamide core structure. Here we describe the structure evolution and biological evaluation of TAS-116. Results: Hit to lead investigations of a 4-(1H-indol-1-yl)benzamide derivative were accomplished by introduction of an alkyl group at the 3-position, inserting one or two nitrogen atoms into the indole ring and ring expansion. As a consequence of chemical structure evolution, we identified novel 5, 6- and 6, 6-fused heteroaromatic rings, especially, the pyrazolopyridine system, which showed increased HSP90-binding affinity in vitro, and cell growth inhibition with HSP90 client degradation. Regarding the 4-position of pyrazolopyridine, substituted imidazoles showed the most favorable profile in vitro inhibitory activities. As for the SAR of the benzamide moiety at the 2- and 3-position, introduction of alkyl and aminoalkyl groups at the 2- or 3-position showed favorable properties, in particular for oral absorption in mice. The clinical candidate, TAS-116, which was identified as a result of these chemistry efforts, showed potent and selective HSP90α/β inhibitory activities in vitro as well as good oral bioavailability in mice, rats and dogs. With regard to potential drug-drug interactions, TAS-116 did not inhibit major cytochrome P450 enzymes in vitro at effective concentrations of TAS-116. TAS-116 demonstrated tumor shrinkage in a NCI-N87 (human gastric cancer) xenograft model without significant body weight loss by oral administration. Preparation of TAS-116 analogues was accomplished through coupling reactions with 4-substituted-pyrazolopyridines which were prepared over several steps and appropriate benzonitriles. Conclusion: A novel, potent and orally active HSP90α/β selective inhibitor, TAS-116, was discovered by rational design and synthesis from a 4-(1H-indol-1-yl)benzamide derivative. The oral clinical candidate, TAS-116, shows favorable drug-like properties and warrants clinical development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C127. Citation Format: Takao Uno, Makoto Kitade, Satoshi Yamashita, Hiromi Oshiumi, Yuichi Kawai, Takashi Mizutani, Chihoko Yoshimura, Yasuo Kodama, Hiromi Muraoka, Kouichi Takahashi, Kazuhiko Yonekura, Shuichi Ohkubo, Teruhiro Utsugi. TAS-116, an orally available HSP90α/β selective inhibitor: Synthesis and biological evaluation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C127.

Abstract C128: TAS-116, an orally available HSP90α/β selective inhibitor, has potent antitumor activity in small cell lung cancer as a single agent and in combination with an anthracycline derivative, amrubicin.

Background: The molecular chaperone HSP90 has been considered a promising target for cancer therapy. We have previously reported the discovery of an orally available HSP90α/β selective inhibitor, TAS-116. TAS-116 led to tumor shrinkage in various human tumor xenograft models, while not inducing retinal toxicity in rats including at doses given above the maximum tolerated dose. Extensive small cell lung cancer (SCLC) is one of the most aggressive types of cancer, and has an unmet medical need for more effective treatments. An anthracycline, amrubicin (AMR), has been used to treat platinum resistant SCLC in Japan. Here we report on the possible application of TAS-116 to treat SCLC both as a single agent and in combination with AMR. Materials and Methods: In vitro drug combination effects were evaluated with calculation of the combination index and cell cycle analysis by fluorescence-activated cell sorting. To clarify the mechanism involved in TAS-116’s enhancement of anthracycline-induced apoptosis the extent of apoptosis was measured in combination with several kinase inhibitors including SB 218078 (CHK1) and MK-2206 (AKT). Antitumor activities of TAS-116 alone and in combination with AMR were evaluated in human SCLC xenograft models. Results: TAS-116 induced cell growth arrest and apoptosis in SCLC cell lines by inhibiting both PI3K/AKT and MAPK signaling through downregulation of HSP90 clients such as AKT and RAF1. TAS-116 also showed antitumor activity against human SCLC tumor xenograft models. In in vitro explorative combination studies in SCLC cell lines, structurally-related anthracyclines, AMR and doxorubicin (DOX), both showed synergistic effect upon apoptosis induction in combination with TAS-116. Moreover, TAS-116 enhanced the antitumor activity of AMR in human SCLC tumor xenograft models. Since anthracyclines exert antitumor activity based on their DNA damaging potential through topoisomerase II inhibition, we evaluated whether DNA damage signaling proteins and apoptosis-related proteins were affected by TAS-116 when given in combination with DOX. TAS-116 significantly reduced phosphorylation of CHK1, AKT and ERK1/2. In combination with these kinase inhibitors, SB 218078 significantly and MK-2206 moderately enhanced DOX-mediated apoptosis. Comparatively, TAS-116 enhanced apoptosis to a greater extent than SB 218078. Conclusion: TAS-116 showed antitumor activity against human SCLC tumor xenograft models alone and in combination with AMR. Our data suggest that TAS-116 enhanced the antitumor activity of anthracyclines through the combined actions of abrogation of the DNA damage-induced G2/M checkpoint and the inhibition of a survival signal by CHK1 and AKT downregulation, respectively. These results suggest that TAS-116 could be a promising agent to treat extensive SCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C128. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Akihiro Hashimoto, Kenjiro Ito, Makoto Kitade, Kazuhiko Yonekura, Shuichi Ohkubo, Teruhiro Utsugi. TAS-116, an orally available HSP90α/β selective inhibitor, has potent antitumor activity in small cell lung cancer as a single agent and in combination with an anthracycline derivative, amrubicin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C128.