Richard Somberg

Overcoming Compound Interference in Fluorescence Polarization-Based Kinase Assays Using Far-Red Tracers

Kinase-mediated phosphorylation of proteins is critical to the regulation of many biological processes, including cell growth, apoptosis, and differentiation. Because of the central role that kinases play in processes that can lead to disease states, the targeting of kinases with small-molecule inhibitors is a validated strategy for therapeutic intervention. Classic methods for assaying kinases include nonhomogenous enzyme-linked immunosorbent assays or scintillation-based formats using [gamma-(32)P]ATP. However, homogenous fluorescence-based assays have gained in popularity in recent years due to decreased costs in reagent usage through miniaturization, increased throughput, and avoidance of regulatory costs associated with the use of radiation. Whereas the readout signal from a nonhomogenous or radioactive assay is largely impervious to interferences from matrix components (such as library compounds), all homogenous fluorescent assay formats are subject to such interferences. Interference from intrinsically fluorescent compounds or from scattered light due to precipitated compounds can interfere with assays that depend on a fluorescence intensity (or fluorescence quenching), fluorescence resonance energy transfer, or fluorescence polarization-based readout. Because these interfering factors show a greater effect at lower wavelengths, one strategy to overcome such interferences is to develop fluorescent assays using longer wavelength (red-shifted) fluorescent probes. In this article, we describe the PanVera PolarScreen far-red fluorescence polarization assay format, which mitigates assay interference from autofluorescent compounds or scattered light through the use of a far-red tracer. The tracer shows substantially less interference from light scatter or autofluorescent library compounds than do fluorescein-based tracers, and gives rise to a larger assay window than the popular far-red fluorophore Cy5.

Fluorescent cascade and direct assays for characterization of RAF signaling pathway inhibitors.

RAF kinases are part of a conserved signaling pathway that impacts cell growth, differentiation, and survival, and RAF pathway dysregulation is an attractive target for therapeutic intervention. We describe two homogeneous fluorescent formats that distinguish RAF pathway inhibitors from direct RAF kinase inhibitors, using B-RAF, B-RAF V599E, and C-RAF. A Förster-resonance energy transfer (FRET) based method was used to develop RAF and MEK cascade assays as well as a direct ERK kinase assay. This method uses a peptide substrate, that is terminally labeled with a FRET-pair of fluorophores, and that is more sensitive to proteolysis relative to the phosphorylated peptide. A second time-resolved FRET-based assay using fluorescently labeled MEK substrate was used to detect direct inhibitors of RAF kinase activity. The cascade assays detect compounds that interact with activated and unactivated kinases within the recapitulated RAF pathway, and the direct assays isolate the point of action for an inhibitor.

Abstract 5532: A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity.

Cell-based assay models are now universally employed as a routine means of establishing and ranking on- and off-target toxicities. It is well appreciated, however, that individual cytotoxic phenotypes are shaped not only by compound dosage, but by the length of compound-cell exposure. In addition to compound-specific attributes, inherent cellular factors such as biotransformation capacity, cell cycle susceptibility, and receptor expression often dictate the kinetics of cytotoxicity. Unfortunately, the majority of conventional cytotoxicity assays are performed at a terminal endpoint (48-72h) which at worst can underestimate cytotoxicity due to biomarker degradation, or at best, fail to reveal important features of the cytotoxic event which may be important for establishing mechanism-of-action. We have developed a pro-fluorescent, cell-impermeant probe which can be applied to cells at the time of dosing to report changes in membrane integrity as they occur in real-time. The dye enters cells with impaired membrane integrity, greatly enhancing fluorescence which is proportional to non-viable cell number. The dye can be used in traditional plate based formats using standard fluorometry or with image based readers (Essen Bioscience Incucyte™) to establish the dose-dependent kinetics of cytotoxicity. Here we describe the dye9s utility using mechanistically distinct cytotoxins with both cancer cell lines (on-target) and terminally differentiated cells (off-target) and correlate this dead cell fluorescence activity to other cytotoxicity biomarkers by same-well multiplexed methods. Citation Format: Richard L. Somberg, Andrew Niles, Tracy Worzella, Dan Lazar. A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5532. doi:10.1158/1538-7445.AM2013-5532

Abstract 3635: Improved T Cell activation bioassays to advance the development of bispecific antibodies and engineered T cell immunotherapies

T cells play a central role in cell-mediated immunity and can mediate long-term, antigen-specific, effector and memory responses. In recent years, a variety of immunotherapy strategies aimed at inducing, strengthening or engineering T cell responses have emerged as promising approaches for the treatment of diseases such as cancer and autoimmunity. Current methods used to measure TCR-mediated T cell proliferation and cytokine production rely on primary PBMCs as a source of T cells, which must be stimulated via co-culture with APCs or anti-TCR/CD3 antibodies. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings. To overcome this barrier, we developed two reporter-based bioluminescent T cell activation bioassays that can be used for the development of bispecific antibodies and engineered T cell immunotherapies. The assays consist of Jurkat T cells genetically engineered to express luciferase downstream of either NFAT or IL-2 response elements. The T cell activation bioassays reflect the mechanisms of action of biologics designed to induce TCR and/or CD28-mediated T cell activation, as demonstrated using anti-CD3 and/or anti-CD28 antibodies as well as blinatumomab, a bispecific antibody that simultaneously binds CD3 expressed on T cells and CD19 expressed on malignant B cells. The bioassays are pre-qualified according to ICH guidelines and show assay specificity, precision, accuracy and linearity required for routine use in potency and stability studies. Finally, our data illustrate the use of reporter-based T cell activation bioassays for characterizing and measuring the activity of engineered chimeric antigen receptor T cells. Citation Format: Richard L. Somberg, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Jey Cheng. Improved T Cell activation bioassays to advance the development of bispecific antibodies and engineered T cell immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3635. doi:10.1158/1538-7445.AM2017-3635

Abstract 360: Detecting intracellular bRAF/cRAF dimerization using a novel luminescent complementation system

BRAF mutations can promote constitutive activation of MEK/ERK signaling, leading to unregulated cell proliferation and tumorigenesis. Although inhibitors targeting oncogenic BRAF have shown promise in preventing cell growth, these same inhibitors can paradoxically activate signaling by inducing BRAF interaction with CRAF. Methods for monitoring this undesired outcome are typically low throughput or use kinase binding domains modified with a membrane targeting sequence. We sought to develop a cell-based assay that could reliably detect dimerization of full-length BRAF/CRAF and could potentially be used to rapidly screen inhibitors for modulation of this interaction. To achieve this objective, we utilized a NanoLuc luciferase-based complementation reporter consisting of 1.3 kD (SmBiT) and 18 kD (LgBiT) fusion tags that reconstitute an active luciferase when brought into proximity. In this optimally configured BRAF/CRAF assay, BRAF-LgBiT and CRAF-SmBiT fusions are stably expressed in mammalian cells, and luminescence, indicative of protein interaction, is monitored using a live cell detection reagent. With this assay, we were able to observe rapid and dose-dependent induction of BRAF/CRAF dimerization in response to a panel of kinase inhibitors. The sensitivity and robustness of the assay permitted reliable screening in both 96- and 384-well formats (Z’ factors > 0.9 and 0.6, respectively). Furthermore, the brightness of the reporter allowed the assay to be monitored in individual cells using bioluminescence imaging. These results suggest this BRAF/CRAF complementation assay can enable rapid, high-throughput profiling of kinase inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway. Citation Format: Richard L. Somberg, Marie K. Schwinn, Michael R. Slater, Thomas Machleidt, Mei Cong, Keith V. Wood. Detecting intracellular bRAF/cRAF dimerization using a novel luminescent complementation system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 360.

Abstract 3883: Miniaturizing the GloSensor™ cAMP assay for MC4R screening using the Echo® acoustic liquid handler

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL As biological assays used for HTS and uHTS are miniaturized, it is critical to maintain assay sensitivity while minimizing reagent volumes and maximizing throughput. The GloSensor™ cAMP Assay provides an extremely sensitive and easy to use, real-time luminescent assay format for the interrogation of over expressed or endogenous GPCRs that signal via changes in the intracellular concentration of cAMP. Tipless, touchless transfers with the Echo® liquid handler eliminate the need for costly disposable tips and greatly simplify assay development efforts. Precise and accurate drop placement eliminates cross-contamination. In this work, we demonstrate optimization of the GloSensor cAMP Assay in low-volume format using a stably transfected HEK293 cell line model expressing the melanocortin 4 receptor (MC4R) and a cAMP-sensing variant of firefly luciferase. We show that the Echo liquid handler is able to titrate small molecule and peptide agonists and antagonists, as well as transfer the GloSensor cAMP Reagent, providing robust assay results that are achieved with significantly reduced volumes of cells and compound. 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 3883. doi:1538-7445.AM2012-3883

Abstract B87: Multiplexed, bioluminogenic probes for evaluating histone deacetylase inhibitor efficacy and safety.

Aberrant epigenetic modifications are known to contribute to hematological and solid cancer malignancies. Members of an important class of chromatin modifying enzymes, histone deacetylases (HDACs), have been found to be dysregulated in many tumor types. Their specific contribution to oncogenesis is unknown, but numerous studies have linked increased HDAC inhibition to transcriptional repression of gene expression, cell cycle checkpoint activation and other tumor suppression activities. Therefore, HDAC inhibitors (HDACi) are an emerging and important class of modulators for use as single agents or in combination with other anti-neoplastics. Many current HDACi have undesirable off-target toxicities, which may be related to inhibition of specific HDAC isozymes that are required for normal cellular function. In an effort to rapidly and efficiently understand the beneficial and detrimental effects of new HDACi within a cellular context, we have developed a set of selective and pan-active bioluminogenic assay probes for evaluating the effects of HDACi. When multiplexed with same well fluorescent viability and cytotoxicity assays, these probes provide a functional readout of HDACi on-target efficacy and off-target safety in primary or cancer cell models. 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 B87.