Starting from thienobenzopyran HTS hit 1, co-crystallization, molecular modeling and metabolic analysis were used to design potent and metabolically stable inhibitors of PI3-kinase. Compound 15 ...demonstrated PI3K pathway suppression in a mouse MCF7 xenograft model.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for ...treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Abstract
Constitutive activation of the RAS, RAF, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) pathway is a common finding in many human cancers. Activating ...mutations in K-RAS or B-RAF constitute over 30% of all mutations in human tumors. It has been demonstrated that RAF inhibitors selectively inhibit B-RAFV600E tumors and not RAS mutant tumors, despite functioning as one of the key effector enzymes downstream of RAS and upstream of MEK. Recent Phase I clinical data with RAF inhibitors has demonstrated a significant response rate (81% response rate) in metastatic melanoma patients with B-RAFV600E-positive tumors (N Engl J Med. 2010 363:809-19). In contrast, the response rates in B-RAFV600E-positive colon tumors have been relatively low. In this work we examine the molecular mechanism of EGFR pathway activation as a mechanism of resistance by comparing the effects of RAF inhibitors against a panel of B-RAFV600E, mutant K-RAS, and WT RAS/RAF tumor lines in the presence and absence of EGF stimulation. We demonstrate that in B-RAF WT lines, pathway induction is observed for RAF inhibitors as evidenced by increases in phospho-MEK and phospho-ERK levels. RAF-inhibitor induced pathway activation in this setting mimics the kinetics and mechanism of EGF-induced pathway stimulation where RAS-GTP levels, C-RAF kinase activity and pERK levels are elevated. Upon stimulation with EGF or serum in B-RAF WT or K-RAS mutant lines, RAF inhibitor induced pathway activation is attenuated. In contrast in B-RAFV600E tumor lines, RAF inhibitors effectively block the MAPK pathway under basal conditions but become ineffective in a subset of B-RAFV600E lines when cells are stimulated with EGF. Additional studies across a broader panel of colon and melanoma mutant B-RAF lines demonstrate that B-RAFV600E lines with high basal levels of EGFR tend to be more resistant to selective B-RAFV600E inhibitors. Immunohistochemistry of colorectal tumor patient samples with either wildtype B-RAF or B-RAFV600E illustrates that colorectal B-RAFV600E tumors are associated with a higher percentage of membrane EGFR. Taken together, these results provide insight into the therapeutic utility of MAPK pathway inhibitors, and emphasize not only the importance of targeting defined genetic backgrounds in cancer treatment but the value in assessing other key pathway markers (such as EGFR) in B-RAFV600E-positive tumor subsets as a factor in sensitivity or resistance to therapy.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 258. doi:10.1158/1538-7445.AM2011-258
The rice Xa21 gene confers resistance to Xanthomonas oryzae pv oryzae in a race-specific manner. Analysis of the inheritance patterns and resistance spectra of transgenic plants carrying six Xa21 ...gene family members indicated that one member, designated Xa21D, displayed a resistance spectrum identical to that observed for Xa21 but conferred only partial resistance. Xa21D encodes a receptor-like protein carrying leucine-rich repeat (LRR) motifs in the presumed extracellular domain. The Xa21D transcript terminates shortly after the stop codon introduced by the retrotransposon Retrofit. Comparison of nucleotide substitutions in the LRR coding regions of Xa21 and Xa21D provided evidence of adaptive selection. Both functional and evolutionary evidence indicates that the Xa21D LRR domain controls race-specific pathogen recognition.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Approximately half of
EGFR
mutant non-small cell lung cancer (NSCLC) patients treated with small molecule EGFR kinase inhibitors develop drug resistance associated with the EGFR T790M “gatekeeper” ...substitution, prompting efforts to develop covalent EGFR inhibitors, which can effectively suppress EGFR T790M in pre-clinical models. However, these inhibitors have yet to prove clinically efficacious, and their toxicity in skin, reflecting activity against wild-type EGFR, may limit dosing required to effectively suppress EGFR T790M
in vivo
. While profiling sensitivity to various kinase inhibitors across a large cancer cell line panel, we identified indolocarbazole compounds, including a clinically well-tolerated FLT3 inhibitor, as potent and reversible inhibitors of EGFR T790M, which spare wild-type EGFR. These findings demonstrate the utility of broad cancer cell profiling of kinase inhibitor efficacy to identify unanticipated novel applications, and they identify indolocarbazole compounds as potentially effective EGFR inhibitors in the context of T790M-mediated drug resistance in NSCLC.
The cloned rice gene, Xa21, confers resistance to multiple pathogen isolates of Xanthomonas oryzae pv. oryzae in transgenic plants. The resistance phenotype was stably transmitted to T1 progeny and ...inherited as a single locus. The T1 progeny were tested for resistance to 32 X. oryzae pv. oryzae isolates from eight countries. Both the engineered line and the donor line showed resistance to 29 isolates and susceptibility to three isolates. The identical resistance spectrum of both lines indicates that the presence of a single member of a multigene family, Xa21, is sufficient to confer multi-isolate resistance. The results presented here have important implications for engineering disease resistance in crop plants
Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the ...C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine ( , ). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody−intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.
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IJS, KILJ, NUK, PNG, UL, UM
The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in ...Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.
Abstract
Constitutive activation of the Ras, RAF, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) pathway is a common finding in many human cancers. Activating ...mutations in K-Ras or B-RAF constitute over 30% of all mutations in human tumors. Therefore, identifying therapeutic strategies to downregulate this pathway could have broad therapeutic impact. It has been demonstrated that RAF inhibitors selectively inhibit B-RAFV600E tumors and not Ras mutant tumors, despite functioning as one of the key effector enzymes downstream of Ras and upstream of MEK. In this work we demonstrate that although RAF inhibitors effectively inhibit phospho-MEK and phospho-ERK levels in B-RAFV600E cells, pathway induction is observed for RAF inhibitors in B-RAF WT/K-Ras Mutant or B-RAF WT/K-Ras WT cell lines as evidenced by increases in phospho-MEK, phospho-ERK and phospho-p90RSK levels. We also show that with increasing concentrations of RAF inhibitors, the downstream pathway can be inhibited in WT B-RAF and mutant K-Ras cell lines. Stimulation of the pathway with growth factors or serum can attenuate phospho-MEK and phospho-ERK induction upon treatment with RAF inhibitors. Additionally, mechanistic studies demonstrate that RAF inhibitors can directly activate RAF kinase activity. Taken together, our data suggests that RAF inhibitors may have opposing functions as both activators and inhibitors of the MAPK pathway, depending on cellular context. These results provide new insight into the therapeutic utility of MAPK pathway inhibitors, and emphasize the importance of correlating target occupancy with pharmacodynamic markers of efficacy, and of targeting defined genetic backgrounds in cancer treatment.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B90.
Abstract
Activating mutations in KRAS or B-RAF are found in over 30% of all human tumors and targeting this pathway could have broad therapeutic impact. Small molecule ATP-competitive RAF kinase ...inhibitors have potent anti-tumor effects on B-RAFV600E tumors but, in contrast to MEK inhibitors, are not potent against RAS mutant tumor models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. In this study we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on cellular context. In B-RAFV600E tumors, RAF inhibitors effectively block the MAPK signaling pathway and decrease tumor growth. Surprisingly, in KRAS mutant and RAS/RAF wildtype tumors, RAF inhibitors activate the RAF/MEK/ERK pathway in a RAS-dependent manner, thus enhancing tumor growth in some xenograft models. Inhibitor binding activates wildtype RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, rather, linked to direct conformational effects of inhibitors on the RAF kinase domain. Based on these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signaling pathways, depending upon cellular context. In addition, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5753.
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