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Two different signaling pathways lead to the activation of the transcription factor NF-κB, initiating distinct biological responses: The canonical NF-κB pathway activation has been implicated in host immunity and inflammatory responses, whereas the noncanonical pathway activation has been involved in lymphoid organ development and B-cell maturation, as well as in the development of chronic inflammatory diseases and some hematologic cancers. The NF-κB-inducing kinase (NIK) is a cytoplasmic Ser/Thr kinase and is a key regulator of the noncanonical pathway. NIK activation results in the processing of the p100 subunit to p52, leading to the formation of the RelB/p52 complex and noncanonical pathway activation. Because of its role in the development of lymphoid malignancies, this kinase has always been considered as an attractive target for the treatment of certain types of cancers and immune diseases. We at Takeda have pursued a drug discovery program to identify small-molecule inhibitors against NIK. This report provides an overview of the data generated from our screening campaign using a small fragment library. Most importantly, we also provide a kinetic analysis of published compounds and chemical series developed at Takeda that are associated with a slow tight-binding mechanism and excellent cellular potency.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

By proposing a quantitative game-theory model of the marriage plot in Jane Austen’s Emma, this essay demonstrates that free-market moral philosophy underwrites Austen’s representation of matrimony and key formal elements of her writing—particularly, matters of verbal profusion. Her famed stylistic “economy” is revealed to be structured by the emerging capitalist economy that Adam Smith theorized in The Wealth of Nations. Establishing the correspondences among several kinds of economy, the essay unites economic and formal approaches to Austen’s work.

We have shown earlier that DNA polymerase beta (Pol beta) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol beta localizes to synapsed axes during zygonema and pachynema, and it associates with the ends of bivalents during late pachynema and diplonema. To test whether these localization patterns reflect a function for Pol beta in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol beta-deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11-dependent gammaH2AX persists on meiotic chromatin, indicating that Pol beta is critical for the repair of SPO11-induced double-strand breaks (DSBs). Pol beta-deficient spermatocytes yielded reduced steady-state levels of the SPO11-oligonucleotide complexes that are formed when SPO11 is removed from the ends of DSBs, and cytological experiments revealed that chromosome-associated foci of replication protein A (RPA), RAD51 and DMC1 are less abundant in Pol beta-deficient spermatocyte nuclei. Localization of Pol beta to meiotic chromosomes requires the formation of SPO11-dependent DSBs. Taken together, these findings strongly indicate that Pol beta is required at a very early step in the processing of meiotic DSBs, at or before the removal of SPO11 from DSB ends and the generation of the 3' single-stranded tails necessary for subsequent strand exchange. The chromosome synapsis defects and Prophase I apoptosis of Pol beta-deficient spermatocytes are likely a direct consequence of these recombination defects.

Mouse lens cultures were employed to study the progression of cataracts initiated by injection of buthionine sulfoximine, an inhibitor of glutathione (GSH) biosynthesis. Culture of lenses removed from untreated mice on postnatal day 7, for 48 hr in the presence of 4 mm BSO, resulted in only limited cataractous changes. To enable substantial progression of cataracts in vitro, it was therefore necessary to pretreat the mice with BSO prior to lens culture. A single injection of BSO (4 nmol mg-1 lens), administered on day 7, resulted in >90% depletion of lens GSH within 3 days, but no visible cataractous changes. The clear lenses were incubated for 29+/-1 hr at 37 degrees C in Medium HL-1, supplemented with EGF, insulin and Ca2+, in the presence or absence of BSO, and were scored for cataract development by previously described criteria. In the absence of BSO, only 4 of 10 lenses developed large opacities. However, in the presence of 4 mm BSO, 40 out of 45 experimental lenses developed opacities affecting at least 50% of the lens visual field and were scored as stages 1C-4, depending upon the extent and density of the cataracts. In addition, three lenses had opacities involving 20-50% of the field (stage 1B). By contrast, less than 10% of lenses from untreated mice incubated in the absence of BSO developed opacities. The cataracts developed in 4 mm BSO were accompanied by reduction of lens glutathione levels to

Optimization of a screening hit from uHTS led to the discovery of TGR5 agonist 32, which was shown to have activity in a rodent model for diabetes.

To compare the effects of two serine-threonine protein kinase inhibitors in a mouse lens culture system previously designed to investigate cortical cataracts caused by L-buthionine sulfoximine (BSO), inhibitor of GSH biosynthesis.Cataract development in HL-1 medium was evaluated visually or by measurement of lens Na+/K+ ratio through atomic absorption. Protein changes were evaluated by 32P-labeling, 2D-gel electrophoresis, phosphorimaging and mass spectrometry. Results. H-7 (50 microM), inhibitor of protein kinase A (PKA) and protein kinase C (PKC), did not cause cataracts, but inhibited BSO cataract development. By contrast, 25 microM H-89, selective inhibitor of PKA, caused large annular cortical cataracts and 100-fold elevation of Na+/K+ within 30 hr in day 10 lenses, in either the presence or absence of BSO. H-89 cataracts were also seen in day 12 and day 21 lenses. 32P-labeling of day 12 lenses pretreated with H-89 displayed more than 80% decrease in phosphorylation of alphaA crystallin, a known substrate of PKA, in the insoluble protein fraction. 2D-gel electrophoresis of day 12 H-89 cataract lens fractions revealed limited degradation of alpha and beta crystallins, degradation of cytoskeletal proteins, and elevated lens Ca2+ (4 nmol/mg wet wt.), suggesting Ca2+-activated proteolysis. Conclusions. High Na+/K+ cataracts are induced by H-89, selective inhibitor of PKA, but not by H-7, an inhibitor of both PKA and PKC that impeded BSO-induced Na+/K+ elevation and cataract. These results suggest contrasting effects of PKA and PKC on lens cation transport and cortical cataract development.

Background: The mammalian target of rapamycin (mTOR), which comprises two protein complexes, TORC1 and TORC2, regulates tumor cell growth, metabolism and motility. In endometrial cancer, the mTOR pathway is aberrantly activated by constitutive mitogen stimuli, such as FGFR2 and multiple genetic mutations such as PIK3CA, PIK3R1/2, PTEN and LKB1. The activation of multiple signaling pathways, cross-feedback and redundancy of these pathways led us to explore the combination of mTOR kinase inhibitor with chemotherapeutic agents for achieving maximal efficacy in preclinical mouse models of endometrial cancer. Results: INK128 is a potent and selective TORC1/2 inhibitor with excellent drug-like properties currently advancing in clinical development. INK128 inhibits endometrial tumor cell proliferation in vitro and displays tumor growth inhibition in endometrial tumor models. In most instances, the anti-tumor activity of INK128 is cytostatic compared to the cytotoxic effect of taxol and carboplatin. In endometrial tumor cells of diverse genetic backgrounds, combining INK128 with taxol resulted in a synergistic inhibition of tumor cell proliferation, sustained suppression of PI3K/AKT/mTOR pathway activity, skewed cell cycle and an increase in apoptosis. INK128 decreased the expression of anti-apoptotic protein MCL-1 and therefore mechanistically enhanced taxol-triggered apoptosis. In endometrial mouse tumor models with PTEN and FGFR2 mutations, the combination induced marked tumor regression and significantly delayed tumor re-growth upon discontinuation of the treatment compared to taxol treatment alone. The combination therapy was well-tolerated and the enhanced anti-tumor efficacy correlated with mechanism-based inhibition of several TORC1 and TORC2 pharmacodynamic markers and induction of apoptosis in vivo. Additionally, combining INK128 with taxol or carbotaxol resulted in tumor regression in taxol-resistant endometrial tumor models. The opportunity of optimizing dose and schedule combination regimens as well as biomarkers predicting sensitivity/resistance to INK128/taxol combination will be discussed. Conclusion: The combination of mTOR kinase inhibitors with chemotherapeutic agents is a compelling strategy to improve therapeutic outcome and to overcome chemo-resistance for tumor types with aberrant activation of the PI3K/mTOR pathway such as endometrial cancer. 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 2745. doi:1538-7445.AM2012-2745

Background: Multiple agents that inhibit angiogenesis and tumor cell growth via the VEGF and mTOR (TORC1) pathways have been approved for locally advanced or metastatic renal cell carcinoma (RCC) in recent years. Despite this progress, improving overall survival remains a significant clinical challenge for the treatment of this cancer type. The tumorigenesis of certain types of RCC is dominated by genes that play critical role in hypoxia response, including stimulation of neo-angiogenesis. Anti-angiogenic therapies target the tumor microenvironment but provide little direct impact to the tumor cell, allowing the potential for disease progression despite treatment. Approval of allosteric partial TORC1 inhibitors in RCC stimulated interest in pursuing TORC1/2 inhibitors, that block mTOR signaling more potently and comprehensively, alone and in combination with anti-angiogenic agents in preclinical models of RCC. Results: INK128 is an orally bioavailable, potent and selective ATP site mTOR kinase inhibitor (TORC1/2) that has entered clinical development. INK128 inhibited the phosphorylation of S6 and 4EBP1, downstream substrates of TORC1, and AKT and NRDG1, downstream substrate of TORC2 in RCC cell lines. TORC1/2 pathway inhibition correlated with potent and complete blockade of cell proliferation. In vivo, daily treatment of INK128 as single agent suppressed tumor growth in multiple mouse RCC xenograft models. Pharmacodynamic analysis demonstrated that INK128 inhibits both TORC1 and TORC2 pathways in vivo. INK128 did not alter the tumor-associated microvasculature density despite significant decrease of VEGF supply by lowering the capacity of protein translation of the tumor cells. This prompted us to explore the potential of the combination of INK128 with anti-angiogenic agents to maximize anti-tumor activity by targeting both tumor microenvironment and tumor cells directly. Co-treatment of INK128 with sorafenib resulted in much enhanced activity in RCC tumor models but did not achieve sufficient tolerability. We next employed an alternating dosing strategy of INK128 and sorafenib to improve tolerability. An optimized dose/schedule sequence of both agents caused sustained inhibition of tumor-associated angiogenesis, suppression of mTOR pathway resulting into marked blockade of tumor growth with significant induction of apoptosis and acceptable tolerability. Conclusion: The sequential, yet potent blockade of both mTOR and VEGF pathways obtained by combining INK128 with sorafenib acts synergistically in inducing cell death and tumor regression in preclinical renal cancer models. Adaptive combination of receptor tyrosine kinase-based anti-angiogenic agents with mTOR kinase inhibitors currently in development may offer a safe and effective therapeutic strategy for renal cancer patients who fail to respond to standard of care therapy. 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 3753. doi:1538-7445.AM2012-3753

Background: The PI3K pathway is one of the most frequently dysregulated pathways in human cancer. Activating mutations in the PIK3CA gene, encoding the p110 catalytic subunit of PI3K, have been identified as a major mechanism of inducing oncogenic PI3K signaling. The high frequency of PIK3CA mutations suggests that PI3K inhibitors may have therapeutic utility in genetically defined tumor populations. The discovery of isoform-selective PI3K inhibitors has proven to be difficult due to the highly homologous nature of all PI3K isoforms and structurally related kinases. PI3Kα-selective inhibitors may permit more potent inhibition of PI3Kα while minimizing side effects and permitting more alternatives for combination therapy relative to non-selective class I PI3K (pan-PI3K) pathway inhibitors, many of which have entered clinical development. Results: Through structure-guided drug design we have discovered INK1117, a novel, potent and selective PI3K inhibitor with good oral bioavailability. INK1117 potently inhibits PI3K and demonstrates a greater than 100-fold selectivity relative to other class I PI3K family members and mTOR as well as a high degree of selectivity against a large panel of protein kinases. INK1117 blocks proliferation of tumor cell lines bearing PIK3CA mutations, and inhibits cellular phosphorylation and activity of AKT. However, INK1117 shows much less activity in PTEN-deficient tumor cells, which typically display constitutive PI3K pathway activation independent of PI3Kα. Daily, oral administration of INK1117 potently inhibits tumor growth in xenograft models bearing PIK3CA oncogenic mutations and, comparable to in vitro studies, INK1117 was not efficacious in tumor models with PTEN and/or KRAS mutations. In contrast to pan-PI3K inhibitors, INK1117 does not induce significant glucose elevation in glucose unchallenged or challenged rodents while levels of circulating Insulin is mildly elevated. The absence of hyperglycemia but presence of moderate hyperinsulinemia in mice treated with INK1117 is in line with results obtained using genetically engineered PI3K deficient mice. Additionally, INK1117 does not significantly impair B and T cell function in vitro and in vivo. INK1117 in combination with targeted agents such as lapatinib or trastuzumab, displayed enhanced activity both in vitro and in vivo. Our preclinical data show that PI3K isoform-selective kinase inhibitors as a single agent can provide comparable or superior efficacy than panPI3K inhibitors in tumors with PIK3CA mutation with better tolerability. The combination of PI3K inhibitors with other oncogene-targeted drugs revealed further improved anti-tumor efficacy. Conclusion: Selective targeting of PI3K combined with companion diagnostic presents a novel therapeutic strategy for patients with PIK3CA mutated cancer types. INK1117 is a potent and orally efficacious PI3Kα-selective kinase inhibitor currently in phase 1 clinical development. 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 A171.

Background: The mammalian target of rapamycin (mTOR) comprises two protein complexes, TORC1 and TORC2, which together regulate cell growth, metabolism, angiogenesis, and cell survival. Because TORC1 and TORC2 play a crucial role in several pathways that are frequently dysregulated in human cancer, the TORC1/2 kinase inhibitors provide a promising class of anti-cancer agents. Activation of multiple genomic and signaling pathways, cross-feedback, pathway redundancy and the capacity for compensatory adaptive response in cancer suggest that the optimal therapeutic effect of mTOR inhibitors will be better exploited by the strategy of rational combination therapies. Results: INK128 is a potent and selective TORC1/2 inhibitor with excellent drug-like properties currently in multiple phase 1 studies. Daily, oral administration of INK128 inhibits tumor growth in multiple xenograft models with predicted pharmacokinetic/pharmacodynamic relationship. In most instances the anti-tumor effect of INK128 is cytostatic compared to the cytotoxic effect of chemotherapeutic agents such as Taxol. To enhance the anti-tumor activity of INK128, we investigated the combination of INK128 with cytotoxic agents such as conventional chemo drugs or apoptosis inducing agents. Treatment of tumors with the cytotoxic agents such as Taxol often leads to activation of the PI3K/AKT/mTOR pathway, which limits the anti-tumor activity of Taxol and may eventually lead to drug resistance. Additionally, Taxol resistance mechanisms include the over-expression of members of the Bcl-2 family of proteins such as MCL-1 and BCL-XL, whose expression is regulated by TORC1 and their function possibly by TORC2. Therefore, there is a strong scientific rationale to investigate the combination of a TORC1/2 inhibitor and Taxol. To this end, we have investigated the preclinical efficacy of INK128 in combination with Taxol in vitro and in vivo. Combining INK128 with Taxol resulted in a synergistic inhibition of tumor cell proliferation and the suppression of PI3K/AKT/mTOR signaling pathways in various cancer types. In most instances alterations in cell cycle and an increase in apoptosis was observed. INK128 decreases the expression of anti-apoptotic protein Mcl-1 and therefore resensitizes Taxol-resistant tumor cells to Mcl-1-dependent build up. In various xenograft models, the combination therapies were well-tolerated and displayed enhanced anti-tumor efficacy, inhibited pharmacodynamic markers, and induced apoptosis. Combination of INK128 with a small molecule inhibitor of Bcl-2 (ABT-263) also led to significant increase of apoptosis in tumor cells but not in normal cells in vitro and enhanced anti-tumor activity in mouse tumor models in vivo. Conclusion: In summary, our data show a potential benefit for combining INK128 with cytotoxic agents. INK128 is currently in a Taxol combination phase Ib study in patients with advanced solid malignancies. 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 A172.