AMPK is a conserved heterotrimeric serine-threonine kinase that regulates anabolic and catabolic pathways in eukaryotes. Its central role in cellular and whole body metabolism makes AMPK a commonly proposed therapeutic target for illnesses characterized by abnormal energy regulation, including cancer and diabetes. Many AMPK modulators, however, produce AMPK-independent effects. To identify drugs that modulate AMPK activity independent of the canonical ATP-binding pocket found throughout the kinome, we designed a robust fluorescence-based high throughput screening assay biased toward the identification of molecules that bind the regulatory region of AMPK through displacement of MANT-ADP, a fluorescent ADP analog. Automated pin tools were used to rapidly transfer small molecules to a low volume assay mixture on 384-well plates. Prior to assay validation, we completed a full assay optimization to maximize the signal-to-background and reduce variability for robust detection of small molecules displacing MANT-ADP. After validation, we screened 13,120 molecules and identified 3 positive hits that dose-dependently inhibited the protein-bound signal of MANT-ADP in the presence of both full-length AMPK and the truncated “regulatory fragment” of AMPK, which is missing the kinase active site. The average Z’-factor for the screen was 0.55 and the compound confirmation rate was 60%. Thus, this fluorescence-based assay may be paired with in vitro kinase assays and cell-based assays to help identify molecules that selectively regulate AMPK with fewer off-target effects on other kinases.
Myoblast proliferation and differentiation are essential for normal skeletal muscle growth and repair. Muscle recovery is dependent on the quiescent population of muscle stem cells - satellite cells. During muscle injury, satellite cells become mitotically active and begin the repair process by fusing with each other and/or with myofibers. Aging, prolonged inactivity, obesity, cachexia and other muscle wasting diseases are associated with a decreased number of quiescent and proliferating satellite cells, which impedes the repair process. A high-content/high-throughput platform was developed and utilized for robust phenotypic evaluation of human primary satellite cells in vitro for the discovery of chemical probes that may improve muscle recovery. A 1600 compound pilot screen was developed using two highly annotated small molecule libraries. This screen yielded 15 dose responsive compounds that increased proliferation rate in satellite cells derived from a single obese human donor. Two of these compounds remained dose responsive when counter-screened in 3-donor obese superlot. The Alk-5 inhibitor LY364947, was used as a positive control for assessing satellite cell proliferation/delayed differentiation. A multivariate approach was utilized for exploratory data analysis to discover proliferation vs. differentiation-dependent changes in cellular phenotype. Initial screening efforts successfully identified a number of phenotypic outcomes that are associated with desired effect of stimulation of proliferation and delayed differentiation.
To evaluate the effects of genetic variations on mRNA splicing, we developed a minigene-based splicing assay using reporter genes encoding luciferase and the multifunctional HaloTag protein. In addition to conventional RT-PCR analysis, splicing events can be monitored in this system using two parameters: luciferase activity and signals derived from HaloTag-containing proteins bound to a fluorescent ligand following SDS-PAGE. The luciferase activity reflects the accumulated amounts of successfully spliced HaloTag-luciferase fusion products, whereas the amounts and sizes of HaloTag-containing proteins provide quantitative insights into precursor, correctly spliced, and aberrantly spliced mRNA species. Preliminary experiments confirmed that the dual reporter minigene assay can provide estimates of overall splicing efficiency based on the levels of protein products. We then used the minigene assay to analyze a case of chronic granulomatous disease that was caused by a G>C mutation at position +5 in the 5'-splice donor site of intron 5 of the CYBB gene. We found that the G>C mutation affected CYBB mRNA splicing by changing a delicate balance of splicing efficiencies of introns 4, 5, and 6.
Microchip electrophoresis (ME) coupled with fluorescence detection was used to estimate the binding activity of aptamer in each systematic evolution of ligands by exponential enrichment (SELEX) round for a target molecule. This approach is a non-radioisotopic, rapid and simple platform, and electrophoretic separation appears to be an effective technique for aptamers of oligonucleotide molecules. We tried to obtain gonadotropin-specific RNA aptamer by the above approach. As a result, the peaks of aptamers based on the conformational differences between them were separated and detected on the electropherograms. Moreover, the intensity of peak of unbound aptamer was decreased with progression through the SELEX rounds, suggesting that RNA aptamer with high affinity was obtained by the proposed method.
Understanding protein function and interaction is central to the elucidation of biological processes. Systematic analysis of protein interactions have shown that the eukaryotic proteome is highly interconnected and that biological function frequently depends on the orchestrated action of many proteins. Perturbation of these functions or interactions can lead to various disease states and pharmacologic intervention can result in corrective therapies. The fact that proteins rarely act in isolation, but rather comprise complex machines that stably and/or transiently interact with many different partners at different times, demands the need for robust tools that allow comprehensive global analyses of these events. Here we describe a powerful protein fusion technology, the HaloTag platform, and how it enables the study of many facets of protein biology by offering a broad choice of applications. We review the development of the key aspects of the technology and it's performance in both in vitro and in vivo applications. In particular, we focus on HaloTag's multifunctional utility in protein imaging, protein isolation and display, and in the study of protein complexes and interactions. We demonstrate it's potential to help elucidate important facets of proteomic biology across complex biological systems at the biochemical, cell-based and whole animal level.
Protocadherin-24 (PCDH24) is linked to the suppression of tumor growth and the inhibition of cell proliferation in the colon cancer cell line HCT116. We previously observed that β-catenin is localized to the plasma membrane when PCDH24 is expressed in these cells, but the molecular mechanisms by which PCDH24 induces the membrane localization of β-catenin remain largely unknown. To clarify these mechanisms, we identified molecules that interact with ectopically expressed PCDH24 in HCT116 cells using a HaloTag® pull-down assay. We found that galectin-1 and galectin-3 physically interact with PCDH24 and are retained at the plasma membrane in association with PCDH24 expression. A luciferase-based pull-down assay using HaloTag-fused galectins revealed that an intracellular region of PCDH24 (amino acids 1186-1280) is essential for this interaction. Furthermore, the over-expression of galectin-1 or -3, or the depletion of endogenous galectins by small interfering RNA modulates β-catenin translocation. We also revealed that the retention of galectin-1 and -3 at the plasma membrane results in the inactivation of PI3K activity. From these findings, we propose a model in which the galectin-anchoring activity of PCDH24 leads to the suppression of β-catenin signaling by the localization of β-catenin at the plasma membrane in PCDH24-expressing HCT116 colon cancer cells.
A hallmark of Huntington's disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins.Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson's, Alzheimer's, and prion diseases.However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear.Identification of small molecule probes that modulate protein aggregation and cytotoxicity caused by aggregated proteins may greatly facilitate the studies on pathogenesis of these diseases and potentially lead to development of new therapies.Based on a detergent insoluble property of the Huntingtin protein aggregates, we have developed a homogenous assay to rapidly quantitate the levels of protein aggregates in a cellular model of Huntington's disease.The protein aggregation assay has also been multiplexed with a protease release assay for the measurement of cytotoxicity resulting from aggregated proteins in the same cells.Through a testing screen of a compound library, we have demonstrated that this multiplexed cytotoxicity and protein aggregation assay has ability to identify active compounds that prevent cell death and/or modulate protein aggregation in cells of the Huntington's disease model.Therefore, this multiplexed screening approach is also useful for development of high-throughput screening assays for other neurodegenerative diseases involving protein aggregation.
The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardisis, resistance to these drugs has been reported and is likely to increase. The Giardia carbamate kinase (glCK) plays an essential role in Giardia metabolism and has no homologs in humans, making it an attractive candidate for anti-Giardia drug development. We have developed a luminescent enzyme coupled assay to measure the activity of glCK by quantitating the amount of ATP produced by the enzyme. This assay is homogeneous and has been miniaturized into a 1536-well plate format. A pilot screen against 4,096 known compounds using this assay yielded a signal-to-basal ratio of 11.5 fold and Z' factor of 0.8 with a hit rate of 0.9 % of inhibitors of glCK. Therefore, this Giardia lamblia carbamate kinase assay is useful for high throughput screening of large compound collection for identification of the inhibitors for drug development.
The patch clamp technique is commonly used in electrophysiological experiments and offers direct insight into ion channel properties through the characterization of ion channel activity. This technique can be used to elucidate the interaction between a drug and a specific ion channel at different conformational states to understand the ion channel modulators' mechanisms. The patch clamp technique is regarded as a gold standard for ion channel research; however, it suffers from low throughput and high personnel costs. In the last decade, the development of several automated electrophysiology platforms has greatly increased the screen throughput of whole cell electrophysiological recordings. New advancements in the automated patch clamp systems have aimed to provide high data quality, high content, and high throughput. However, due to the limitations noted above, automated patch clamp systems are not capable of replacing manual patch clamp systems in ion channel research. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and differentiated cells that derive from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Therefore, further improvements in flexibility with regard to cell types and data quality will broaden the applications of the automated patch clamp systems in both academia and industry.
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.
Technological and methodological advances have been critical for the rapidly evolving field of proteomics. The development of fusion tag systems is essential for purification and analysis of recombinant proteins. The HaloTag is a 34 KDa monomeric protein derived from a bacterial haloalkane dehalogenase. The majority of fusion tags in use today utilize a reversible binding interaction with a specific ligand. The HaloTag system is unique in that it forms a covalent linkage to its chloroalkane ligand. This linkage permits attachment of the HaloTag to a variety of functional reporters, which can be used to label and immobilize recombinant proteins. The success rate for HaloTag expression of soluble proteins is very high and comparable to maltose binding protein (MBP) tag. Furthermore, cleavage of the HaloTag does not result in protein insolubility that often is observed with the MBP tag. In the present report, we describe applications of the HaloTag system in our ongoing investigation of protein-protein interactions of the Y. pestis Type 3 secretion system on a custom protein microarray. We also describe the utilization of affinity purification/mass spectroscopy (AP/MS) to evaluate the utility of the Halo Tag system to characterize DNA binding activity and protein specificity.
The function of a particular protein is dependent upon its localization and milieu. The ability to track the "fate" of a protein is a valuable tool to elucidate its function. We present the use of HaloTag technology to study the localization and fate of human Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9).The role of PCSK9 in the regulation of circulating low density lipoprotein-cholesterol (LDL-c) levels is ascribed to binding of circulating PCSK9 to the LDL receptor (LDLR) and subsequent lysosomal degradation of LDLR. However, hints in the literature indicate that intracellular PCSK9 may act on the LDLR, possibly during processing of newly synthesized protein. To address this question, the source and fate of intracellular PCSK9 requires further investigation.We applied HaloTag technology to distinguish the source of intracellular PCSK9 and showed that newly synthesized intracellular PCSK9 has unique localization from the PCSK9 after re-uptake. This suggests different functions of PCSK9 while interacting with the LDLR.
Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.
From in vitro proteomic protein analysis to in vivo animal models, various protein tagging systems are designed to provide useful functionalities. Commonly used affinity tags for protein purification and capture include GST, c-myc, metal ion affinity tag His6Tag, immuno-affinity FLAG tag, and maltose binding protein (MBP) tag [1-4]. For cellular imaging analysis, fluorescent proteins have served as markers for protein localization, translocation, interactions, and conformational change [5, 6]. However, in addition to the limited functionality, these protein fusion tags are further limited in that they cannot be used for in vivo animal models. Instead, bioluminescence, near-infrared (NIR) /IR800, Mag-netic Resonance Imaging (MRI), positron emission tomography (PET), and single-photon emission tomography (SPECT) are used to detect protein markers in vivo [7, 8]. Due to limited capabilities for individual tags, studies involving multiple analytical methods often require the use of more than one tag and multiple genetic constructions. HaloTag® technology provides an efficient way to use a single tag readily reconfigured to meet the needs of different applications, from in vitro protein purification to cellular protein complex analysis, and in animal models. SNAP-tag and CLIP-tag provide similar synthetic fluorophores with switchable spectroscopic properties to visualize biochemical activities in living cells [9, 10]. The HaloTag® technology possesses two distinct moieties: the HaloTag® reporter protein and the HaloTag ligand. The HaloTag reporter protein is fused to the N- or C-terminus of a protein of interest (POI). The reporter is a monomeric, 34 KDa engineered protein developed from a rare bacterial hydrolase. There is no endogenous equivalent in eukaryotic cells, and like most bacterial reporter/tag systems, it has extremely low background. In this special issue, Dr. Encell et al. [11] describe the design and molecular evolution of the HaloTag® reporter system. Using directed mutagenesis, the binding kinetics was significantly improved for the initial version of the reporter HaloTag2 by opening up the chloroalkane binding pocket. Random mutagenesis was then used to further evolve HaloTag® together with its linker sequence into a more soluble and stable variant, HaloTag7, which is suitable for applications across mammalian and bacterial cells as well as in vitro. The chloroalkane ligand is the second component in the HaloTag® technology platform and it can covalently bind to HaloTag® protein. Chloroalkane ligands can carry many different functional groups including i) solid surface for protein immobilization and display that can be used for protein purification, interaction analysis and display of antigens; and ii) fluorescent dyes for protein quantification, gel labeling, and cellular imaging analysis. Also in this special edition, Drs. Urh and Rosenberg [12] provide a comprehensive review of the HaloTag® technology platform and its wide range of applications both in vitro and in vivo. By comparing the performance of direct protein purification using different tags with 20 randomly selected ORFs encoded in the genome of S. pneumonia, Peterson and Kwon [13] demonstrated that HaloTag significantly enhances expression and solubility of target proteins. Furthermore, they presented a successful case study of using a HaloTag® fusion protein to identify protein-protein and protein-DNA interactions in Y. pestis Type 3 secretion system. Unlike other fluorescence protein reporters where a separate construct needs to be made for each “color” of fluorescence, HaloTag® and HaloTag®-POI fusions are not fluorescent until the addition of a fluorescent HaloTag ligand. This important distinction allows for spatial and temporal control of labeling, and hence renders HaloTag an ideal technology for cellular imaging [14]. Ai et al. [15] exploited the advantages of the unique property of ligand interchangeability and demonstrated the localization and fate of human Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9), a protein important in regulating circulating low density lipoprotein-cholesterol (LDL-c) levels. After imaging confirmation for proper localization and pharmacological behavior, the same construct can also be used for protein pull-down analysis for novel protein complex study using HaloLink resin. Indeed Ose et al. [16] demonstrated that galectin-1 and galectin-3 physically interact with ectopically expressed PCDH24- HaloTag® in HCT116 cells using a HaloTag® pull-down assay. The study supports a model that the galectin-anchoring activity of PCDH24 leads to the suppression of β-catenin signaling by the localization of β-catenin at the plasma membrane in PCDH24-expressing HCT116 colon cancer cells. The irreversible, covalent bond formed between HaloTag ligand and reporter protein is in stoichiometric (1:1) ratio. Fluorescence intensities of protein bands following SDS-PAGE likely reflect the relative levels of HaloTag proteins after cell lysis. Oshima et al. [17] combined the advantages of HaloTag and the Dual Luciferase Assay to quantitatively examine splice variants (i.e., unspliced, aberrantly spliced, and correctly) of mRNA products to determine relative splicing efficiencies with and without mutations represented in a case of chronic granulomatous disease that was caused by a G→C mutation at position +5 in the 5’-splice donor site of intron 5 of the CYBB gene. The advantages of the multifunctional nature of HaloTag® system do not stop at translating in vitro proteomics into cellular analysis. Recently, several papers have clearly demonstrated its utility in animal imaging using HaloTag MRI and PET ligands [18, 19]. Tseng et al. [20] spatially evaluated the in vivo application using a HaloTag® HCT116 tumor cell line overexpressing xenograft model and showed that flourophore-conjugated ligands are well tolerated after parenteral injection and exhibit enhanced tumor uptake for non-invasive in vivo imaging. Recent studies from Dr. Crew’s lab have provided an exciting new application of the HaloTag technology wherein labeling HaloTag® proteins with low-molecular-weight hydrophobic molecules (HyT) lead to targeted proteasomal degradation [21]. With tools for targeted genome engineering, we can i) integrate HaloTag® into endogenous genes of interest and study the localization of POI at endogenous levels using a variety of HaloTag® fluorescence ligands at cellular level; ii) pull down HaloTag®-POI using HaloTag® resin for protein complex analysis; and iii) apply HyT to knock down HaloTag®-POI at physiological level for functional analysis. The same concept can be applied to in vivo animal models by generating knock in HaloTag® mice. Development of HaloTag®-MRI and HaloTag®-PET ligands in concert with HyT will provide researchers integrated tools for studying drug targets in both cellular and animal models.
The enzymes that regulate histone methylation states and the protein domains that recognize methylated histone residues have been implicated in a number of human diseases, including cancer, as a result of their ability to affect transcriptional changes by altering chromatin structure. These proteins are recognized as potential therapeutic targets for the treatment of diseases associated with epigenetic disruption; however, few inhibitors of their activity have been identified. The majority of histone demethylase and methyltransferase enzyme inhibitors have been discovered on the basis of their structural similarity to substrates or known inhibitors of enzymes with analogous mechanisms. The general lack of potency and specificity of these compounds indicates that novel chemotypes are needed to address the large number of recently discovered histone-modifying enzymes. High-throughput screening (HTS) allows rapid testing of chemically diverse small molecule libraries, provided assays amenable to HTS exist. Here we review the biochemical and cellular assays available for testing the proteins and enzymes that regulate histone methylation. Progress in the development of high-throughput, sensitive, and robust assays will enable discovery of small molecules for epigenetic therapy.
The recognition of methyl-lysine and -arginine residues on both histone and other proteins by specific “reader” elements is important for chromatin regulation, gene expression, and control of cell-cycle progression. Recently the crucial role of these reader proteins in cancer development and dedifferentiation has emerged, owing to the increased interest among the scientific community. The methyl-lysine and -arginine readers are a large and very diverse set of effector proteins and targeting them with small molecule probes in drug discovery will inevitably require a detailed understanding of their structural biology and mechanism of binding. In the following review, the critical elements of methyl-lysine and -arginine recognition will be summarized with respect to each protein family and initial results in assay development, probe design, and drug discovery will be highlighted.
CPT-11 is a widely-used anti-cancer drug that is converted in vivo to its active metabolite, SN-38. In the liver, enzymes detoxify SN-38 by coupling it to a glucuronidate moiety and this inactive compound (SN-38G) is excreted into the gastrointestinal tract. In the intestine, commensal bacteria convert the SN-38G back to the active and toxic SN-38 using bacterial β-glucuronidase enzyme (GUS). This intestinal SN-38 causes debilitating diarrhea that prevents dose-intensification and efficacy in a significant fraction of patients undergoing CPT-11 treatment for cancer. This CPT-11 metabolic pathway suggests that small molecule inhibitors of GUS may have utility as novel therapeutics for prevention of dose-limiting diarrhea resulting from CPT-11 therapy. To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library. Novel inhibitors of GUS were identified with IC50 values ranging from 50 nM to 4.8 µM. These compounds may be useful as chemical probes for use in proof-of-concept experiments designed to determine the efficacy of GUS inhibitors in altering the intestinal metabolism of drugs. Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.
There are about fifty SET domain protein methyltransferases (PMTs) in the human genome, that transfer a methyl group from S-adenosyl-L-methionine (SAM) to substrate lysines on histone tails or other peptides. A number of structures in complex with cofactor, substrate, or inhibitors revealed the mechanisms of substrate recognition, methylation state specificity, and chemical inhibition. Based on these structures, we review the structural chemistry of SET domain PMTs, and propose general concepts towards the development of selective inhibitors.
Growing evidence suggests that protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) are associated with the development of various human diseases, including cancer, inflammation, and psychiatric disorders. Given the significant role of these proteins in human disease, efforts to discover selective small-molecule inhibitors of these enzymes are quickly gaining momentum. In this review, we focus on the recent progress in the discovery of selective PKMT and PRMT inhibitors. A future perspective on developing methyltransferase inhibitors is also offered.
Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.
