Rachel Friedman Ohana

HaloTag: a novel protein labeling technology for cell imaging and protein analysis.

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.

HaloTag7: a genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification.

Over-expression and purification of soluble and functional proteins remain critical challenges for many aspects of biomolecular research. To address this, we have developed a novel protein tag, HaloTag7, engineered to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. HaloTag7 was designed to bind rapidly and covalently with a unique synthetic linker to achieve an essentially irreversible attachment. The synthetic linker may be attached to a variety of entities such as fluorescent dyes and solid supports, permitting labeling of fusion proteins in cell lysates for expression screening, and efficient capture of fusion proteins onto a purification resin. The combination of covalent capture with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags and enables efficient capture even at low expression levels. Following immobilization on the resin, the protein of interest is released by cleavage at an optimized TEV protease recognition site, leaving HaloTag7 bound to the resin and pure protein in solution. Evaluation of HaloTag7 for expression of 23 human proteins in Escherichia coli relative to MBP, GST and His(6)Tag revealed that 74% of the proteins were produced in soluble form when fused to HaloTag7 compared to 52%, 39% and 22%, respectively, for the other tags. Using a subset of the test panel, more proteins fused to HaloTag7 were successfully purified than with the other tags, and these proteins were of higher yield and purity.

Target engagement and drug residence time can be observed in living cells with BRET

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.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

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.

HaloTag-based purification of functional human kinases from mammalian cells.

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.

Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntingtons disease.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement

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.

Purification of Recombinant Proteins from Cultured Mammalian Cells by HaloTag Technology.

Cultured mammalian cells provide an environment ideal for producing functional recombinant mammalian proteins. However, low expression levels of recombinant proteins present a challenge for their detection and purification. This unit will focus on HaloTag, a protein fusion tag designed to bind selectively and covalently to a chloroalkane ligand that may be attached to a variety of functional groups, allowing both protein detection and immobilization. Detection of HaloTag‐fusion protein is achieved through binding to a fluorescent chloroalkane ligand, enabling rapid optimization of expression levels. HaloTag‐based purification uses covalent capture of the HaloTag fusion onto HaloLink resin coupled with proteolytic cleavage to release the protein of interest from the resin. Covalent binding provides efficient protein capture regardless of expression level and eliminates protein loss during washes of the resin and as a result, offers significant improvements in protein recovery and purity over traditional non‐covalent approaches.

Target Identification Using Cell Permeable and Cleavable Chloroalkane Derivatized Small Molecules

An important aspect for gaining functional insight into the activity of small molecules revealed through phenotypic screening is the identification of their interacting proteins. Yet, isolating and validating these interacting proteins remains difficult. Here, we present a new approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules to isolate their respective protein targets. Derivatization of small molecules with the CA moiety has been shown to not significantly impact their cell permeability or potency, allowing for phenotypic validation of the derivatized small molecule prior to capture. The retention of cell permeability also allows for treatment of live cells with the derivatized small molecule and the CA moiety enables rapid covalent capture onto HaloTag coated magnetic beads. Additionally, several options are available for the elution of interacting proteins, including chemical cleavage of the CA moiety, competitive elution using excess unmodified small molecule, or sodium dodecyl sulfate (SDS) elution. These features taken together yield a highly robust and efficient process for target identification, including capture of weak or low abundance interactors.

Abstract C158: A chemoproteomics strategy for target identification and lead compound optimization using chloroalkane derivatized compounds

The Identification and validation of drug targets is an industry wide challenge. There is a very clear and urgent need for technologies that can identify the interaction partners of small molecules. Chemical proteomics is one technology that has attracted attention as a solution to the drug target identification problem. Here we present a new approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules to isolate their respective protein partners. In general derivatization of small molecules with the CA moiety does not impact their cell permeability and has limited impact on potency, allowing for phenotypic assays of the derivatized compound to be performed. The retention of cell permeability also allows for the capture process to be performed from live cells treated with the CA-compound. This process is also compatible with competition assays, which can be used to evaluate and compare other compounds exhibiting a similar mode of action. Here we present a study using Dasatinib-CA, a modified kinase inhibitor and potent anti-cancer drug which binds to a broad range of kinases. First we performed target enrichment experiments by incubating K562 cells with the modified Dasatinib (Dasatinib-CA). Cells were lysed and the Dasatinib-CA, together with the bound targets, was rapidly captured onto magnetic resin coated with HaloTag. Unmodified compound was used to competitively elute putative interacting proteins. Secondly using the same assay format we evaluated the relative target affinities of Dasatinib-CA versus competing molecules. Competition assays were performed by incubating multiple mixtures of Dasatinib analogues and Dasatinib-CA at varying relative concentrations. Eluted proteins were processed using SDS-PAGE and in-gel digestion. For target identification experiments peptides were analyzed using label free mass spectrometry. For the competition assays digested material was labeled with Tandem Mass Tags (TMT) 10plex reagents. Peptides were analyzed using nanoscale LC-MS/MS coupled with a Q Exactive mass spectrometer (Thermo). Protein identification and quantitation was performed with MaxQuant (MaxQuant.org) and data validation and visualization was performed using Perseus (Perseus-framework.org). Using this approach we identified over 30 kinases, including known membrane associated and membrane protein targets. This work presented here highlights a new method for chemical proteomics and demonstrates utility of the platform to enable target identification and to evaluate competitor molecules. Citation Format: Michael Ford, Richard Jones, Ravi Amunugama, Danette Daniels, Rachel Ohana, Sergiy Levin, Thomas Kirkland, Marjeta Urh, Keith Wood. A chemoproteomics strategy for target identification and lead compound optimization using chloroalkane derivatized compounds. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C158.