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RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF signalosome in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular selectivity of clinical-stage drugs for individual protomers within BRAF, CRAF, and ARAF heterodimers in complex with mutant KRAS-GTP revealed that ARAF protomer-engagement, but not engagement of BRAF or CRAF is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and highlight the avoidance of ARAF protomers for a cohort of RAF inhibitors undergoing clinical evaluation.

Current small molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket, exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the switch-II pocket is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a novel cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the switch-II pockets of many GTP hydrolysis-deficient KRAS hotspot (G12, G13, Q61) mutants are accessible using non-covalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the switch-II pocket as a privileged drug binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of neurodegenerative disorders. SW-100 (1a), a phenylhydroxamate-based HDAC6 inhibitor (HDAC6i) bearing a tetrahydroquinoline (THQ) capping group, is a highly potent and selective HDAC6i that was shown to be effective in mouse models of Fragile X syndrome and Charcot-Marie-Tooth disease type 2A (CMT2A). In this study, we report the discovery of a new THQ-capped HDAC6i, termed SW-101 (1s), that possesses excellent HDAC6 potency and selectivity, together with markedly improved metabolic stability and druglike properties compared to SW-100 (1a). X-ray crystallography data reveal the molecular basis of HDAC6 inhibition by SW-101 (1s). Importantly, we demonstrate that SW-101 (1s) treatment elevates the impaired level of acetylated α-tubulin in the distal sciatic nerve, counteracts progressive motor dysfunction, and ameliorates neuropathic symptoms in a CMT2A mouse model bearing mutant MFN2. Taken together, these results bode well for the further development of SW-101 (1s) as a disease-modifying HDAC6i.

Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.

Concerted multidisciplinary efforts have led to the development of Cyclin-Dependent Kinase inhibitors (CDKi’s) as small molecule drugs and chemical probes of intracellular CDK function. However, conflicting data has been reported on the inhibitory potency of CDKi’s and a systematic characterization of affinity and selectivity against intracellular CDKs is lacking. We have developed a panel of cell-permeable energy transfer probes to quantify target occupancy for all 21 human CDKs in live cells, and present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi’s and tool molecules. We observed unexpected intracellular activity profiles for a number of CDKi’s, offering avenues for repurposing of highly potent molecules as probes for previously unreported targets. Overall, we provide a broadly applicable method for evaluating the selectivity of CDK inhibitors in living cells, and present a refined set of tool molecules to study CDK function., Cyclin-dependent kinase (CDK) inhibitors are widely used both in the clinic and for basic research aimed at dissecting the specific cellular functions of specific CDKs. Here, the authors report the development of a panel of fluorescent reporter probes and provide a comprehensive profile of the inhibitory activity of several CDK inhibitors towards all 21 CDKs in living cells.

Fc gamma receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of monoclonal antibody (mAb) therapeutics and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, immunoglobulin G (IgG) immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signalling pathway of the inhibitory FcγRIIB (CD32B), utilised NanoLuc® Binary Interaction Technology (NanoBiT™) to measure recruitment of the Src homology 2 (SH2) domain-containing inositol phosphatase 1 (SHIP-1) to the immunoreceptor tyrosine-based inhibitory motif (ITIM) of this receptor. Such recruitment required prior crosslinking of an immunoreceptor tyrosine-based activation motif (ITAM)-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signalling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, Rituximab:anti-idiotype complexes and anti-trinitrophenol (TNP)-TNP complexes in a sensitive manner (≤1µg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.

A panel of cell-permeable energy transfer probes has been developed to quantify target occupancy for all 21 CDKs in live, intact cells. Here we present the first comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi’s and tool molecules. Here we provide a broadly applicable method for evaluating the selectivity of chemical matter for CDKs in living cells, and present a refined set of tool molecules to study CDK function.

Isoxazole is a five-membered heterocycle that is widely used in drug discovery endeavors. Here, we report the design, synthesis, and structural and biological characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of the active-site zinc ion that differs from the preferred monodentate coordination observed for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These findings suggest that the antitumor activity of SS-208 is mainly mediated by immune-related antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC50 shifts...

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that hijack ubiquitin E3 ligases and induce degradation of intracellular proteins through a tightly regulated proteosomal mechanism. Although several successful PROTACs have been developed against key intracellular target classes including bromodomains, kinases, and nuclear hormone receptors, these bivalent molecules often suffer from poor cell permeability due to high molecular weight. To enable a high-throughput, quantitative readout for PROTAC permeability and E3 ligase occupancy in living cells, we have developed a panel of target engagement (TE) assays for key E3 ligase including CRBN, VHL, XIAP, cIAP, and MDM2. These intracellular assays are the first biophysical method to enable the quantitative determination of compound occupancy, potency, and residence time for specific target proteins inside living cells. The assays provide a direct measure of compound binding or occupancy to a target under physiological conditions using bioluminescent resonance energy transfer (BRET). Citation Format: James Vasta, Cesear Corona, Jennifer Wilkinson, Morgan R. Ingold, Chad Zimprich, Marie Schwinn, Thomas Machleidt, Jim Hartnett, Mei Cong, Frank Fan, Richard L. Somberg, Matthew Robers. A live cell method to assess E3 ligase and target protein occupancy for PROTACs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6407.

Cyclin-dependent kinases (CDK) play key roles in diverse cellular functions including cell cycle control, cell proliferation, and transcriptional regulation. Due to these important roles, CDKs have been targeted for cancer therapeutic development, which has resulted in hundreds of small molecule inhibitors. However, conflicting data of CDK inhibitor potency has been reported and a rigorous comparison of inhibitor affinity and selectivity for intracellular CDKs is lacking. To address this need, we have developed a panel of cell-permeable energy transfer probes or tracers to enable comprehensive quantitation of inhibitor target occupancy and affinity for CDKs in live cells via a bioluminescent energy transfer (BRET) method. Specifically, the quantitative BRET-based capability is achieved via energy transfer from cell-permeable fluorescent tracers reversibly engaged to NanoLuc luciferase-tagged CDK proteins expressed in live cells. An untagged cyclin can be introduced into the cells at the same time as the NanoLuc-CDK, allowing for the interrogation of a specific CDK-cyclin pair. The BRET-based method can be used in equilibrium binding analysis. In addition, time-dependent target-compound occupancy (or residence time) can also be obtained with this method. We will present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of clinically-advanced CDK inhibitors and chemical probes across the CDK family. We observed unexpected intracellular activity profiles for some clinically-advanced CDK inhibitors. We further evaluated mechanisms for achieving target selectivity through target residence time under non-equilibrium cell culture conditions. This BRET-based method is broadly applicable for evaluating the occupancy, affinity, and selectivity of chemical matter for CDKs in live cells. Citation Format: James Vasta, Carrow Wells, Cesear Corona, Jennifer Wilkinson, Chad Zimprich, Morgan Ingold, Domenic Ogno, Marie Schwinn, Thomas Machleidt, Mei Cong, Frank Fan, Kristin Huwiler, Amy Landreman, Julie E. Pickett, Kilian Huber, Matthew Robers. Examination of clinically relevant inhibitor selectivity in live cells across the CDK family [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5808.

Disease-modifying therapies are needed for Fragile X Syndrome (FXS), as at present there are no effective treatments or cures. Herein, we report on a tetrahydroquinoline-based selective histone deacetylase 6 (HDAC6) inhibitor SW-100, its pharmacological and ADMET properties, and its ability to improve upon memory performance in a mouse model of FXS, Fmr1-/- mice. This small molecule demonstrates good brain penetrance, low-nanomolar potency for the inhibition of HDAC6 (IC50 = 2.3 nM), with at least a thousand-fold selectivity over all other class I, II, and IV HDAC isoforms. Moreover, through its inhibition of the α-tubulin deacetylase domain of HDAC6 (CD2), in cells SW-100 upregulates α-tubulin acetylation with no effect on histone acetylation and selectively restores the impaired acetylated α-tubulin levels in the hippocampus of Fmr1-/- mice. Lastly, SW-100 ameliorates several memory and learning impairments in Fmr1-/- mice, thus modeling the intellectual deficiencies associated with FXS, and hence providing a strong rationale for pursuing HDAC6-based therapies for the treatment of this rare disease. ispartof: ACS CHEMICAL NEUROSCIENCE vol:10 issue:3 pages:1679-1695 ispartof: location:United States status: published

Summary For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose., Graphical Abstract, Highlights • The approach enables quantitative profiling of 178 full-length kinases • Compared with biochemical approaches, this is a better predictor of cellular potency • An unexpected intracellular selectivity is observed for certain kinase inhibitors • A mechanistic analysis of ATP interference on target engagement is performed, Vasta et al. describe a broad-spectrum approach (BRET) to quantitatively measure target engagement for kinases inside live cells. Compared with biochemical measurements, the analysis revealed an improved intracellular selectivity profile for clinically relevant kinase inhibitors. This serves as a mechanistic tool to determine the effect of cellular ATP on engagement potency.

Background Stem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity. New method In this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPC Luc2 ) in the rat striatum long-term. Results We show that hNPC Luc2 are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPC Luc2 contralateral migration. Comparison with existing methods Some of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood–brain barrier remains intact. Conclusions These new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment., Drug molecules operate through physical interaction with specific cellular targets, and understanding this interaction is important for mechanisms and the potential therapeutic effect of drug candidates. Here, the authors show that bioluminescence resonance energy transfer can be used to monitor the intracellular engagement of a drug with its target.

Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies – Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR) – we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be beneficial for screening a large number of antibody samples during early monoclonal development phase.

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.

Chromobacterium violaceum was recovered at necropsy from the lungs, liver, spleen, and an interscapular abscess of a Chinese red panda (strain 98-9187) [J. Vet. Diagn. Invest. 12 (2000) 177]. As the lungs exhibited extensive, necrotizing lesions harboring bacterial aggregates, we sought to determine whether C. violaceum produced an elastase that might in part account for these lesions. The C. violaceum type strain (ATCC 12472 T ) and strain 98-9187 were shown to exhibit elastolytic activity by elastin Congo red and elastin nutrient agar assays. The activity was isolated from the periplasmic fraction and was present throughout the growth cycle. Activity increased markedly in late logarithmic phase growth. In elastin-limiting medium, activity rapidly decreased in early stationary phase indicating a tight regulation of yield. The activity was optimal at neutral pH and was sensitive to the metalloproteinase inhibitors EDTA and 1,10-phenanthroline. Activity was restored upon addition of zinc indicating the enzyme is a zinc metalloproteinase. A band corresponding to purified elastase activity was present at ∼30 kDa in a denaturing polyacrylamide gel.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.

We present a biophysical method to directly measure target engagement within intact mammalian cells using bioluminescence energy transfer (BRET). Compound interactions with intracellular targets can be detected with complete specificity by their ability to compete with energy transfer complexes introduced into the cells. These complexes can be detected at physiologically relevant levels by exploiting an extraordinarily bright luciferase (NanoLuc), together with fluorescent tracers optimized for cell-permeability and spectral resolution from the luciferase. We demonstrate applications of the technology for target engagement among key drug target classes, including; kinases, histone deacetylases (HDACs), bromodomains, and the methyltransferase EZH2. Intracellular selectivity and affinity profiles of various reference compounds and approved drugs will be presented. For a panel of HDAC inhibitors, affinity profiles for specific HDAC isozymes strongly correlate with phenotypic potencies (e.g. cell viability). Furthermore, the luminescent output of the energy transfer complex enables a technique to monitor ligand occupancy in real-time. Association and dissociation rates can be derived from the kinetic measurements, providing a means to quantify drug residence time on select targets within intact cell populations. This novel application of intracellular BRET should significantly advance target engagement work flows, and allow for intracellular target affinities to be coupled to phenotypic outcomes. Citation Format: Matthew B. Robers, Melanie Dart, Chad Zimprich, Thomas Kirkland, Sergiy Levin, Thomas Machleidt, Jim Hartnett, Kris Zimmerman, Rachel Ohana, Danette Daniels, Mei Cong, Frank Fan, Keith Wood. Measuring intracellular target engagement and drug residence time with nanoBRET. [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 3512. doi:10.1158/1538-7445.AM2015-3512

Microtissues produced in 3D cell culture are much more representative of actual living tissue compared to monolayers produced in 2D cell culture. In fact, in the area of oncology research, multicellular tumor spheroids are considered an excellent platform for testing drug delivery and efficacy. As the necessity for established 3D cell culture models rises, there is also a need for convenient assays that have been specifically demonstrated to be effective for use with 3D microtissues. The more complex architecture of 3D microtissues demands increased lytic effectiveness and reagent penetration, characteristics that are often only minor considerations for reagents designed for 2D cell culture. Here we report on a variety of bioluminescent and fluorescent cell-based assays applied to hanging-drop spheroids produced from HCT116 colon cancer cells. The first assay to be described is an ATP detection reagent for measuring cell viability. This reagent has both an improved formulation and an optimized assay protocol and has clear advantages over other viability assays. Other cell health assays will also be described, including reagents that measure cell death, apoptosis, mechanistic cytotoxicity, or reporter gene expression. These additional assays do not require a change in formulation, but do require new protocols in order to optimize their effectiveness when applied to 3D microtissues. As with their application to cells in 2D culture, these “add-mix-measure” reagents are robust and amenable to both low- and high-throughput applications. Citation Format: Michael P. Valley, Kevin R. Kupcho, Chad A. Zimprich, Andrew L. Niles, James J. Cali, Jens M. Kelm, Wolfgang Moritz, Dan F. Lazar. Luminescent cell health assays for tumor spheroid evaluation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3731. doi:10.1158/1538-7445.AM2014-3731

September, 2014 The use of 3D cell cultures continues to emerge as improved in vitro models that are more physiologically relevant and more predictive of cytotoxic events in whole animals. Cells cultured in 3D models often acquire large in vivo-like structures compared to the thickness of a 2D monolayer of cells grown on standard plastic plates. Multicellular 3D culture systems containing more than one cell type and exhibiting formation of a complex extracellular matrix provide a challenge for assay chemistries originally designed for measuring events from monolayers of cells. There is an unmet need for guidelines for design and verification of assays effective for larger 3D microtissues. Critical factors to consider for each model system include effective penetration of detection reagents and/or complete lysis of cells using combinations of detergent and physical disruption. We will present the results of experiments designed to improve performance of assays for 3D spheroids. We have improved the lytic capacity of assay reagents by optimizing the detergent concentration and the parameters for physical disruption necessary to extract the desired markers. Improved protocols and reagents will be described to measure ATP as a cell viability marker, caspase-3/7 activity as an apoptosis marker, glutathione as a marker of oxidative stress and HIF-1 promoter driven expression of luciferase as a hypoxia marker.

Phenotypic-based screening of small molecule libraries is a significant trend in drug discovery. However, deciphering the intracellular interactions between proteins and small molecules that mediate phenotypic outcomes remains a major challenge. Current approaches rely on linking the small molecule to a surface or affinity tag (e.g., biotin), allowing selective capture of the corresponding protein target for identification by mass spectrometry. Verifying that the linkage method does not disrupt the pharmacological activity is important for ensuring maintenance of the binding interaction with the intracellular targets. Consequently, although target capture is commonly done from cell lysates, methods compatible with living cells are preferable. We have developed such a method based on a novel chloroalkane tag that minimally affects compound potency and cell permeability. This allows phenotypic outcomes to be recapitulated by the modified compound, increasing confidence in the biological relevance of the captured proteins. The tagged compound is allowed to bind at equilibrium to intracellular targets, then cells are lysed and the chloroalkane bound to its protein targets is quickly captured onto magnetic particles containing immobilized HaloTag. The rapid isolation method minimizes complex collapse, preserves low affinity interactions, and in combination with low background adsorption facilitates target identification by mass spectrometry. Engagement of identified targets by compounds in living cells can be verified using bioluminescence energy transfer (BRET). NanoLuc luciferase fused to the target protein serves as a BRET donor to the compound derivatized with fluorophore adducts. As a model to test this combined target capture/target validation work flow we selected the interaction of histone deacetylase (HDAC's) and Vorinostat (SAHA) a broad- HDAC inhibitor. This family of deacetylases exhibits a range of cellular abundance and affinity to SAHA (0.001-1µM), and has members localized in the nucleus or cytoplasm. Results: Treatment of K562 cells with SAHA or a derivative having the chloroalkane tag revealed high cellular potency against HDAC class I/IIb with IC50 of 0.1µM and 0.2µM respectively. These results indicate minimal impact of the chloroalkane modification on the drug potency. For target identification, the SAHA chloroalkane derivative was allowed to bind at equilibrium to intracellular targets followed by rapid capture onto immobilized HaloTag. By this method we were able to identify all the direct interactors of SAHA (i.e., HDAC 1,2,3,6 and 8) regardless of their subcellular localization, abundance, or affinity. The intracellular BRET approach was next used to validate the set of identified HDAC's and to rank order their relative binding affinity to SAHA. Our results suggested that these approaches used in combination provide reliable identification of targets in living cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C224. Citation Format: Rachel Friedman Ohana, Matt Robers, Thomas A. Kirkland, Carolyn C. Woodroofe, Chad Zimprich, Tetsuo H. Uyeda, Paul Otto, Sergiy Levin, Keith Wood. Identification of drug targets via their intracellular interactions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C224.

It has been well demonstrated that the morphology, gene expression, and overall biological response of cells assessed in 3-dimensional (3D) culture models are frequently more physiologically relevant than that of cells studied in standard 2D culture formats. In spite of rapidly growing interest, one hindrance to the use of 3D models for drug discovery and other research efforts is the lack of convenient and effective assays explicitly validated for application to 3D microtissues. Here we report on a bioluminescent ATP-detection assay comprised of both an improved formulation and an optimized protocol specifically designed to measure the viability of cells grown in 3D culture. The ability of the 3D-optimized reagent to effectively lyse cells from throughout the full thickness of 3D microtissues, including cancer cell spheroids, was clearly demonstrated by use of confocal laser fluorescent microscopy in conjunction with a fluorogenic, membrane-impermeant DNA-binding dye. Importantly, the assay's ability to accurately report the ATP content, and therefore viability, of various 3D microtissues (including a range of cancer cell line spheroids) was demonstrated by comparison to a trichloroacetic acid ATP extraction method. In scaffold-free as well as scaffold-dependent 3D models, recovery and detection of greater than 80% of ATP present was routinely observed. In contrast, another frequently used ATP-detection assay was found to have greatly reduced lytic and ATP detection properties ( Citation Format: Michael P. Valley, Chad Zimprich, James J. Cali, Dan F. Lazar. A bioluminescent cell viability assay optimized for 3D microtissues. [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 5531. doi:10.1158/1538-7445.AM2013-5531

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

Background The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag®, to the extracellular domain of a truncated integrin. Results Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture. Conclusion The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells.