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Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson’s disease (PD). Pathogenic variants in LRRK2, including the common variant G2019S, result in increased LRRK2 kinase activity, supporting the therapeutic potential of LRRK2 kinase inhibitors for PD. To better understand the role of LRRK2 in disease and to support the clinical development of LRRK2 inhibitors, quantitative and high-throughput assays to measure LRRK2 levels and activity are needed. We developed and applied such assays to measure the levels of LRRK2 as well as the phosphorylation of LRRK2 itself or one of its substrates, Rab10 (pT73 Rab10). We observed increased LRRK2 activity in various cellular models of disease, including iPSC-derived microglia, as well as in human subjects carrying the disease-linked variant LRRK2 G2019S. Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk. Finally, we optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic.

Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson’s disease (PD). Pathogenic variants in LRRK2, including the common variant G2019S, result in increased LRRK2 kinase activity, supporting the therapeutic potential of LRRK2 kinase inhibitors for PD. To better understand the role of LRRK2 in disease and to support the clinical development of LRRK2 inhibitors, quantitative and high-throughput assays to measure LRRK2 levels and activity are needed. We developed and applied such assays to measure the levels of LRRK2 as well as the phosphorylation of LRRK2 itself or one of its substrates, Rab10 (pT73 Rab10). We observed increased LRRK2 activity in various cellular models of disease, including iPSC-derived microglia, as well as in human subjects carrying disease-linked variant in LRRK2 (G2019S). Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk. Finally, we optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic.

Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies. This article describes a procedure for treating cells and preparing cell lysates, as well as a procedure for generating RPPAs using these lysates. A method for probing, imaging, and analyzing RPPAs is also described. These procedures are readily adaptable to a wide range of studies of cell signaling in response to drugs and other perturbations. © 2016 by John Wiley & Sons, Inc.

Kaposi's sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.

The in-cell western (ICW) technique is a cell-based immunoassay method for quantitative measurement of protein expression or phosphorylation levels that can be used for both small molecule and siRNA screening. The method involves growth of cells in microplates, fixation, permeabilization, and staining with specific antibodies and/or cell labeling dyes. ICW assays take advantage of the properties of near-infrared dyes to achieve higher signal-to-noise ratios than are possible for methods utilizing fluorophores in the visible range of the spectrum, and typically involve measurements using two fluorescent channels: one to measure levels of the target of interest, and one to measure total cell number for normalization. The ICW method is readily adaptable to high-throughput format and has been successfully used with a variety of targets and cell lines. The protocols in this unit describe an ICW procedure for quantitative measurement of rpS6-phosphorylation as an endpoint for monitoring mTORC1 signaling in HeLa cells. This assay can be used for small molecule or siRNA screening, and with modification is adaptable to other cell lines and targets. Curr. Protoc. Chem. Biol. 3:39-52 © 2011 by John Wiley & Sons, Inc. Keywords: in-cell western; ICW; high-throughput screening; mTOR; rpS6; rapamycin; phosphorylation

Binding of eIF4E to the cap structure (m(7)GpppN) plays a critical role in mRNA translation. To study the interaction between eIF4E and cap, and to identify small molecule inhibitors of their binding, we synthesized a fluorescent-labeled cap analogue and used it to develop a fluorescence-polarization assay. This preliminary communication describes the synthesis of a fluorescein labeled 7-methylguanosinemonophosphate, and its dose dependent binding to purified human eIF4E as demonstrated by the fluorescence polarization assay.

The eukaryotic initiation factor 4G (eIF4G) is the core of a multicomponent switch controlling gene expression at the level of translation initiation. It interacts with the small ribosomal subunit interacting protein, eIF3, and the eIF4E/cap-mRNA complex in order to load the ribosome onto mRNA during cap-dependent translation. We describe the solution structure of the complex between yeast eIF4E/cap and eIF4G (393–490). Binding triggers a coupled folding transition of eIF4G (393–490) and the eIF4E N terminus resulting in a molecular bracelet whereby eIF4G (393–490) forms a right-handed helical ring that wraps around the N terminus of eIF4E. Cofolding allosterically enhances association of eIF4E with the cap and is required for maintenance of optimal growth and polysome distributions in vivo. Our data explain how mRNA, eIF4E, and eIF4G exists as a stable mRNP that may facilitate multiple rounds of ribosomal loading during translation initiation, a key determinant in the overall rate of protein synthesis.

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML-RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.

The technique of fluorescence polarization (FP) is based on the observation that when a fluorescently labeled molecule is excited by polarized light, it emits light with a degree of polarization that is inversely proportional to the rate of molecular rotation. This property of fluorescence can be used to measure the interaction of a small labeled ligand with a larger protein and provides a basis for direct and competition binding assays. FP assays are readily adaptable to a high-throughput format, have been used successfully in screens directed against a wide range of targets, and are particularly valuable in screening for inhibitors of protein-protein and protein-nucleic acid interactions when a small binding epitope can be identified for one of the partners. The protocols in this article describe a general procedure for development of FP assays to monitor binding of such a peptide or oligonucleotide to a protein of interest. Curr. Protoc. Chem Biol. 1:1-15. © 2009 by John Wiley & Sons, Inc.

Cancer cells can be drug resistant due to genetic variation at multiple steps in the drug response pathway, including drug efflux pumping, target mutation and blunted apoptotic response. These are not discriminated by conventional cell survival assays. Here, we report a rapid and convenient high content cell-imaging assay that measures multiple physiological changes in cells responding to anti-mitotic small-molecule drugs. Our one-step, no-wash assay uses three dyes to stain living cells and is much more accurate for scoring weakly adherent mitotic and apoptotic cells than conventional antibody-based assays. We profiled responses of 33 cell lines to 8 anti-mitotic drugs at multiple concentrations and time points using this assay, and deposited our data and assay protocols into a public database (http://lincs.hms.harvard.edu/). Our data discriminated between alternative mechanisms that compromise drug sensitivity to Paclitaxel, and revealed an unexpected bell-shaped dose-response curve for BI2536, a highly selective inhibitor of Polo-like kinases. Our approach can be generalized, is scalable and should therefore facilitate identification of molecular biomarkers for mechanisms of drug insensitivity in high-throughput screens and other assays.

International audience; Considerable evidence indicates that the general blockade of protein synthesis prevents both the initial consolidation and the postretrieval reconsolidation of long-term memories. These findings come largely from studies of drugs that block ribosomal function, so as to globally interfere with both cap-dependent and -independent forms of translation. Here we show that intra-amygdala microinfusions of 4EGI-1, a small molecule inhibitor of cap-dependent translation that selectively disrupts the interaction between eukaryotic initiation factors (eIF) 4E and 4G, attenuates fear memory consolidation but not reconsolidation. Using a combination of behavioral and biochemical techniques, we provide both in vitro and in vivo evidence that the eIF4E-eIF4G complex is more stringently required for plasticity induced by initial learning than for that triggered by reactivation of an existing memory.

The mTORC1 pathway is a central regulator of cell growth, and defective mTORC1 regulation plays a causative role in a variety of human diseases, including cancer, tumor syndromes such as the tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), and metabolic diseases such as diabetes and obesity. Given the importance of mTORC1 signaling in these diseases, there has been significant interest in developing screening methods suitable for identifying inhibitors of mTORC1 activation. To this end, we have developed a high-throughput, cell-based assay for the detection of rpS6-phosphorylation as a measure of mTORC1 signaling. This assay takes advantage of the “In Cell Western” (ICW) technique using the Aerius infrared imaging system (LI-COR® Biosciences). The ICW procedure involves fixation and immunostaining of cells in a manner similar to standard immunofluorescence methods but takes advantage of secondary antibodies conjugated to infrared-excitable fluorophores for quantitative detection by the Aerius® scanner. In addition, the cells are stained with an infrared-excitable succinimidyl ester dye, which covalently modifies free amine groups in fixed cells and provides a quantitative measure of cell number. We present validation data and pilot screens in a 384-well format demonstrating that this assay provides a statistically robust method for both small molecule and siRNA screening approaches designed to identify inhibitors of mTORC1 signaling.

Treatment of BRAFV600E melanomas with drugs, such as vemurafenib, that inhibit RAF/MEK signaling is effective in the short term, but remission is not durable. Acquired drug resistance is thought to involve short-term adaptive responses that compensate for RAF/MEK inhibition via up-regulation of other pro-growth mechanisms. Thus, understanding and ultimately preventing adaptive responses is a key to durable therapy. Systematic data comparing BRAFV600E tumor cells is generally lacking and it is not known whether adaptation is fundamentally similar across cell types or among individual cells within a cell population. We apply a systematic approach to studying the responses of human melanoma cell lines to five drugs, RAF and MEK inhibitors, with the overall goal of (i) characterizing variability in adaptation with time, dose, cell type and across individual cells, (ii) discovering new or poorly characterized adaptive mechanisms, and (iii) demonstrating the effectiveness of a high-throughput approach involving multiplex measurement, single-cell analysis and computational modeling. The data involves time-course measurement of total level and activity of signaling proteins and cell state markers using array-based methods and single-cell immunofluorescence assays as well as measurement of apoptosis and cell viability under the same conditions. Statistical modeling using partial least squares regression (PLSR) revealed which of the changes in the ∼200,000 point dataset were phenotypically consequential. We found that responses to RAF inhibitors are remarkably diverse and involve multiple pathways that can be up or down-regulated over time, with significant variability across cell types and individual cells. We identified a role for JNK/c-Jun signaling in altering the cell-cycle distribution of melanoma cells, causing apoptosis-resistant cells to accumulate and drug maximal effect (Emax) to fall; co-drugging with RAF and JNK inhibitors or JUN knockdown reverse this effect. The primary effect of JNK inhibitors is to minimize the cell-to-cell variability in pS6 suppression, promoting the induction of apoptosis. Our study shows that a systems-level approach (combining high density time-dependent measurements, quantitative modeling and single-cell analysis) may provide a general framework for evaluating new drugs with adaptive and paradoxical response, and identifying potentially useful combination therapies. Citation Format: Mohammad Fallahi-Sichani, Nathan J. Moerke, Mario Niepel, Tnghu Zhang, Nathanael S. Gray, Peter K. Sorger. Single-cell analysis of adaptive resistance and fractional responses of melanoma cells to RAF/MEK inhibition. [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 3744. doi:10.1158/1538-7445.AM2015-3744

International audience; Assembly of the eIF4E/eIF4G complex has a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the 4E-BPs, which compete with eIF4G for binding to eIF4E and which have tumor-suppressor activity. To pharmacologically mimic 4E-BP function we developed a high-throughput screening assay for identifying small-molecule inhibitors of the eIF4E/eIF4G interaction. The most potent compound identified, 4EGI-1, binds eIF4E, disrupts eIF4E/eIF4G association, and inhibits cap-dependent translation but not initiation factor-independent translation. While 4EGI-1 displaces eIF4G from eIF4E, it effectively enhances 4E-BP1 association both in vitro and in cells. 4EGI-1 inhibits cellular expression of oncogenic proteins encoded by weak mRNAs, exhibits activity against multiple cancer cell lines, and appears to have a preferential effect on transformed versus nontransformed cells. The identification of this compound provides a new tool for studying translational control and establishes a possible new strategy for cancer therapy.

The cap-binding protein eIF4E is the first in a chain of translation initiation factors that recruit 40S ribosomal subunits to the 5' end of eukaryotic mRNA. During cap-dependent translation, this protein binds to the 5'-terminal m(7)Gppp cap of the mRNA, as well as to the adaptor protein eIF4G. The latter then interacts with small ribosomal subunit-bound proteins, thereby promoting the mRNA recruitment process. Here, we show apo-eIF4E to be a protein that contains extensive unstructured regions, which are induced to fold upon recognition of the cap structure. Binding of eIF4G to apo-eIF4E likewise induces folding of the protein into a state that is similar to, but not identical with, that of cap-bound eIF4E. At the same time, binding of each of the binding partners of eIF4E modulates the kinetics with which it interacts with the other partner. We present structural, kinetic and mutagenesis data that allow us to deduce some of the detailed folding transitions that take place during the eIF4E interactions.

Global conformational transitions are of central functional importance for many enzymes and binding proteins. It is not known, however, how much variability can exist in such structural-functional linkages. We have characterized the global magnitude of the T to R conformational transition of Escherichia coli aspartate transcarbamylase (ATCase) by measuring (1) hydration changes using osmotic stress and (2) hydrodynamic changes using high-precision analytical gel chromatography. We find that specific mutations can alter the structural magnitude of the enzyme's conformational transition without abolishing allostery, suggesting that some degree of plasticity exists in the conformational component of allostery.

Melanoma is the deadliest form of skin cancer and the leading cause of death from skin disease. The BRAF(V600E) mutation is found in approximately 60% of melanomas. It causes constitutive activation of the RAF/MEK/ERK signaling pathway in these tumors leading to uncontrolled cell growth. Inhibition of BRAF(V600E) induces apoptosis in melanoma cells which has been used as the basis for molecular targeted therapy of melanoma. Small-molecule compounds such as Vemurafenib, Dabrafenib and other selective BRAF(V600E) inhibitors specifically bind BRAF(V600E), and have been shown to cause dramatic, but transient, tumor regression in vivo. The precise mechanisms of cell death following BRAF inhibition remain obscure; the literature posits a diversity of ways in which apoptosis is induced. However, it is unclear whether a single but complex network is involved or whether mechanisms differ from one cell type to the next. This is an important issue because use of BRAF(V600E) inhibitors in the treatment of melanoma is seriously challenged by the absence of durable responses; even in initially responsive patients tumors usually re-emerge within months and are observed to be drug resistant. An important challenge is thus to understand determinants of initial sensitivity to BRAF inhibition beyond V600E mutation and to determine which of these are involved in resistance. Using high throughput measurement of the abundance and state of modification of multiple proteins representing diverse signaling pathways at the basal level and over time in multiple melanoma cell lines exposed to different doses of BRAF inhibitors and data-driven mathematical modeling, we determine how the complex dynamics of signaling pathways are controlled in different cell lines and how drugs targeting BRAF(V600E) alter this signaling. Our analysis shows that the basal level of some proteins such as PTEN, pERK, pS6, pAkt, and β-catenin vary up to 100-fold among different lines prior drug treatment. Further, these cell lines show different degrees of oncogene addiction and remarkable diversity with respect to the level of ERK inhibition and apoptosis as well as activation or inhibition of other signaling cascades following BRAF inhibition. We use mathematical modeling to relate basal levels of signaling proteins and their dynamics following BRAF inhibition to pharmacological descriptors of sensitivity to multiple BRAF drugs. Thus, we identify key signaling nodes within the signaling networks of the melanoma cells which determine the differential phenotypic consequences of BRAF inhibition. This approach will be critical for predicting and ultimately improving drug sensitivity in resistant tumors (e.g. via co-treatment strategies). Citation Format: Mohammad Fallahi-Sichani, Nathan J. Moerke, Anahita Dastur, Cyril H. Benes, Peter K. Sorger. A systems biology approach to understanding differential phenotypic outcome of BRAF(V600E) inhibition in melanoma cells. [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 5213. doi:10.1158/1538-7445.AM2013-5213