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Liu Q.,Harvard University | Xu C.,Ludwig Center at Dana Farber Harvard Cancer Center | Kirubakaran S.,Harvard University | Zhang X.,Harvard University | And 27 more authors.
Cancer Research | Year: 2013

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1- dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases includingATM(EC50, 28 nmol/L), ATR (EC50, 35 nmol/L), and DNA-PK (EC50, 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal- regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings wheremTOR signaling has a pathogenic role. © 2013 American Association for Cancer Research.


Lin E.C.K.,ActivX Biosciences Inc. | Hu Y.,ActivX Biosciences Inc. | Amantea C.M.,ActivX Biosciences Inc. | Pham L.M.,ActivX Biosciences Inc. | And 9 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2012

AX10185, the phenyl amide of xanthurenic acid, was found to be a sub-100 nM inhibitor of Lp-PLA 2. However, in the presence of EDTA the inhibitory activity of AX10185 was extinguished while the enzymatic activity of Lp-PLA 2 did not change. Subsequent metal screening experiments determined the inhibition to be Zn 2+ dependent. Structure-activity relationship studies indicated the presence of the 4-hydroxy group to be critical and selected substituted phenyl, polycyclic, and cycloaliphatic amides of xanthurenic acid to be well tolerated. © 2011 Elsevier Ltd. All rights reserved.


Rosenblum J.S.,ActivX Biosciences Inc. | Nomanbhoy T.K.,ActivX Biosciences Inc. | Kozarich J.W.,ActivX Biosciences Inc.
FEBS Letters | Year: 2013

The largest mammalian enzyme family is the kinases. Kinases and other nucleotide-binding proteins are key regulators of signal transduction pathways and the mutation or overexpression of these proteins is often the difference between health and disease. As a result, a massive research effort has focused on understanding how these proteins function and how to inhibit them for therapeutic benefit. Recent advances in chemical biological tools have enabled functional interrogation of these enzymes to provide a deeper understanding of their physiological roles. In addition, these innovative platforms have paved the way for a new generation of drugs whose properties have been guided by functional profiling. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.


Vetter M.L.,Harvard University | Rodgers M.A.,Harvard University | Rodgers M.A.,University of Southern California | Patricelli M.P.,ActivX Biosciences Inc. | Yang P.L.,Harvard University
ACS Chemical Biology | Year: 2012

Many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used RNA- and protein-based profiling methods that monitor steady-state gene expression. Mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. Taking dengue virus 2 (DV2) infection as a model system, we utilized reactive ATP- and ADP-acyl phosphates as chemical proteomic probes to detect changes in host kinase function that occur within the first hour of infection. The DNA-dependent protein kinase (DNA-PK) was discovered as a host enzyme with significantly elevated probe labeling within 60 min of DV2 infection. Increased probe labeling was associated with increased DNA-PK activity in nuclear lysates and localization of DNA-PK in nucleoli. These effects on DNA-PK were found to require a postfusion step of DV2 entry and were recapitulated by transfection of cells with RNA corresponding to stem loop B of the DV2 5′ untranslated region. Upon investigation of the potential downstream consequences of these phenomena, we detected a modest but significant reduction in the interferon response induced by DV2 in cells partially depleted of the Ku80 subunit of DNA-PK. These findings identify changes in DNA-PK localization and activity as very early markers of DV2 infection. More broadly, these results highlight the utility of chemoproteomic profiling as a tool to detect changes in protein function associated with different cell states and that may occur on very short time scales. © 2012 American Chemical Society.


Okerberg E.S.,ActivX Biosciences Inc. | Brown H.E.,ActivX Biosciences Inc. | Minimo L.,ActivX Biosciences Inc. | Alemayehu S.,ActivX Biosciences Inc. | And 4 more authors.
Journal of the American Chemical Society | Year: 2014

Here we describe a chemical proteomics strategy using ATP acyl phosphates to measure the formation of a protein:protein complex between p38α and mapkap kinases 2 and/or 3. Formation of the protein:protein complex results in a new probe labeling site on p38α that can be used to quantify the extent of interaction in cell lysates and the equilibrium binding constant for the interaction in vitro. We demonstrate through RNA interference that the labeling site is dependent on formation of the protein:protein complex in cells. Further, we identify that active-site-directed, small-molecule inhibitors of MK2/3 selectively inhibit the heterodimer-dependent probe labeling, whereas p38α inhibitors do not. These findings afford a new method to evaluate p38α and MK2/3 inhibitors within native biological systems and a new tool for improved understanding of p38α signaling pathways. © 2014 American Chemical Society.


Li B.,ActivX Biosciences Inc. | Cociorva O.M.,ActivX Biosciences Inc. | Nomanbhoy T.,ActivX Biosciences Inc. | Weissig H.,ActivX Biosciences Inc. | And 11 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2013

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC50 = 160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC50 = 47 nM) was a highly specific JNK inhibitor. © 2013 Elsevier Ltd. All rights reserved.


Seto S.,Kyorin Pharmaceutical Co. | Yumoto K.,Kyorin Pharmaceutical Co. | Okada K.,Kyorin Pharmaceutical Co. | Asahina Y.,Kyorin Pharmaceutical Co. | And 6 more authors.
Bioorganic and Medicinal Chemistry | Year: 2012

The design, synthesis, and evaluation of 6-6-7 tricyclic quinolones containing the strained spirocycle moiety aiming at the GSK-3β inhibitor were described. Among the synthesized compounds, 44, having a cyclobutane ring on a spirocycle, showed excellent GSK-3β inhibitory activity in both cell-free and cell-based assays (IC 50 = 36 nM, EC 50 = 3.2 μM, respectively). Additionally, 44 decreased the plasma glucose concentration dose-dependently after an oral glucose tolerance test in mice. © 2011 Elsevier Ltd. All rights reserved.


Trademark
ActivX Biosciences Inc. | Date: 2011-07-05

Diagnostic preparations for scientific research use; reagents for scientific research use; assays and reagents for genetic research; reagents for use in the field of activity-based proteomics.


Trademark
ActivX Biosciences Inc. | Date: 2011-06-28

Medical products, namely probes.


Trademark
ActivX Biosciences Inc. | Date: 2010-01-11

Medical products, namely, probes.

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