High-throughput FRET Research Articles

Progress toward high-throughput single-molecule FRET mapping of the sequence landscape of RNA structure and dynamics

Progress toward high-throughput single-molecule FRET mapping of the sequence landscape of RNA structure and dynamics

From thiol-subtilisin to omniligase: Design and structure of a broadly applicable peptide ligase

Omniligase-1 is a broadly applicable enzyme for peptide bond formation between an activated acyl donor peptide and a non-protected acyl acceptor peptide. The enzyme is derived from an earlier subtilisin variant called peptiligase by several rounds of protein engineering aimed at increasing synthetic yields and substrate range. To examine the contribution of individual mutations on S/H ratio and substrate scope in peptide synthesis, we selected peptiligase variant M222P/L217H as a starting enzyme and introduced successive mutations. Mutation A225N in the S1′ pocket and F189W of the S2′ pocket increased the synthesis to hydrolysis (S/H) ratio and overall coupling efficiency, whereas the I107V mutation was added to S4 pocket to increase the reaction rate. The final omniligase variants appeared to have a very broad substrate range, coupling more than 250 peptides in a 400-member library of acyl acceptors, as indicated by a high-throughput FRET assay. Crystal structures and computational modelling could rationalize the exceptional properties of omniligase-1 in peptide synthesis

Actin-binding compounds, previously discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

Actin's interactions with myosin and other actin-binding proteins are essential for cellular viability in numerous cell types, including muscle. In a previous high-throughput time-resolved FRET (TR-FRET) screen, we identified a class of compounds that bind to actin and affect actomyosin structure and function. For clinical utility, it is highly desirable to identify compounds that affect skeletal and cardiac muscle differently. Because actin is more highly conserved than myosin and most other muscle proteins, most such efforts have not targeted actin. Nevertheless, in the current study, we tested the specificity of the previously discovered actin-binding compounds for effects on skeletal and cardiac α-actins as well as on skeletal and cardiac myofibrils. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that several of these effects were different for skeletal and cardiac actin isoforms. We also found that several of these compounds affected ATPase activity differently in skeletal and cardiac myofibrils. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

High-Throughput FRET Screening in Living Cells Based on Lifetime Detection to Identify Small-Molecule Effectors of Serca

The development of an effective high-throughput screening (HTS) paradigm using FRET biosensors in living cells has enabled us to identify multiple classes of small-molecules that alter the activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). We aim to discover small-molecule activators that improve SERCA's function for the treatment of heart failure. We have validated an intramolecular SERCA FRET biosensor (red and green fluorescent proteins fused to distinct SERCA domains) by screening a small test library using novel plate readers (PRs) that detect the fluorescence lifetime (FLT) (Schaaf et al, 2017). Here we describe an FLT-PR that scans a 1536-well plate in ≤2.5 min. This instrument is equipped with multichannel FLT detection at two emission wavelengths, used to filter out fluorescent compounds that would be falsely identified as hits. False hits were further eliminated using FRET biosensors based on non-specific peptide linkers, and using spectral analysis. FRET biosensors were developed and tested based on two different isoforms of SERCA: the SERC2a cardiac-specific isoform and SERCA2b non-muscle isoform were used to discover compounds with specificity toward SERCA2a. Concentration-response studies were performed to determine potency of compounds. Hits were assessed by two separate functional assays to determine how they alter ATPase activity and calcium-uptake. The majority were found to be inhibitors, but some were activators, and some compounds affected the coupling of SERCA's ATPase and calcium transport activities Our future focus is to evaluate the structure-function relationships of the newly discovered SERCA activators, using medicinal chemistry to optimize drug-like properties. These new HTS methods are being applied to a wide range of drug targets.

Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains

β-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal β-1,4-linked galactose and α-2,6-linked sialic acid to N-glycans. Their exclusive interaction in the Golgi compartment is a prerequisite for their full catalytic activity, whereas a lack of this interaction is associated with cancers and hypoxia. To date, no structural information exists that shows how glycosyltransferases functionally assemble with each other. Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and high-throughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding. Moreover, we found that the assembly of ST6GAL1 homomers in the endoplasmic reticulum before ST6GAL1 activation in the Golgi utilizes the same noncatalytic surface, whereas B4GALT1 uses its active-site surface for assembly, which silences its catalytic activity. Last, we show that the homomeric and heteromeric B4GALT1/ST6GAL1 complexes can assemble laterally in the Golgi membranes without forming cross-cisternal contacts between enzyme molecules residing in the opposite membranes of each Golgi cisterna. Our results provide detailed mechanistic insights into the regulation of glycosyltransferase interactions, the transitions between B4GALT1 and ST6GAL1 homo- and heteromers in the Golgi, and cooperative B4GALT1/ST6GAL1 function in N-glycan synthesis.

Nonconventional glucagon and GLP-1 receptor agonist and antagonist interplay at the GLP-1 receptor revealed in high-throughput FRET assays for cAMP

G protein-coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively. However, glucagon secreted from pancreatic α-cells may accumulate at high concentrations to exert promiscuous effects at the β-cell GLP-1R, as may occur in the volume-restricted microenvironment of the islets of Langerhans. Furthermore, systemic administration of GluR or GLP-1R agonists and antagonists at high doses may lead to off-target effects at other receptors. Here, we used molecular modeling to evaluate data derived from FRET assays that detect cAMP as a read-out for GluR and GLP-1R activation. This analysis established that glucagon is a nonconventional GLP-1R agonist, an effect inhibited by the GLP-1R orthosteric antagonist exendin(9-39) (Ex(9-39)). The GluR allosteric inhibitors LY2409021 and MK 0893 antagonized glucagon and GLP-1 action at the GLP-1R, whereas des-His1-[Glu9]glucagon antagonized glucagon action at the GluR, while having minimal inhibitory action versus glucagon or GLP-1 at the GLP-1R. When testing Ex(9-39) in combination with des-His1-[Glu9]glucagon in INS-1 832/13 cells, we validated a dual agonist action of glucagon at the GluR and GLP-1R. Hybrid peptide GGP817 containing glucagon fused to a fragment of peptide YY (PYY) acted as a triagonist at the GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). Collectively, these findings provide a new triagonist strategy with which to target the GluR, GLP-1R, and NPY2R. They also provide an impetus to reevaluate prior studies in which GluR and GLP-1R agonists and antagonists were assumed not to exert promiscuous actions at other GPCRs.

N-acetylglucosaminyltransferases and nucleotide sugar transporters form multi-enzyme\u2013multi-transporter assemblies in golgi membranes in vivo

Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin-myosin structure and function. Actin-myosin interactions play crucial roles in the generation of cellular force and movement. Numerous mutations and post-translational modifications of actin or myosin disrupt muscle function and cause life-threatening syndromes. Here, we used a FRET biosensor to identify modulators that bind to the actin-myosin interface and alter the structural dynamics of this complex. We attached a fluorescent donor to actin at Cys-374 and a nonfluorescent acceptor to a peptide containing the 12 N-terminal amino acids of the long isoform of skeletal muscle myosin's essential light chain. The binding site on actin of this acceptor-labeled peptide (ANT) overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-ANT complex as in the actin-myosin complex and (b) a significant decrease in actin-ANT FRET upon binding myosin. A high-throughput FRET screen of a small-molecule library (NCC, 727 compounds), using a unique fluorescence lifetime readout with unprecedented speed and precision, showed that FRET is significantly affected by 10 compounds in the micromolar range. Most of these "hits" alter actin-activated myosin ATPase and affect the microsecond dynamics of actin detected by transient phosphorescence anisotropy. We conclude that the actin-ANT TR-FRET assay enables detection of pharmacologically active compounds that affect actin structural dynamics and actomyosin function. This assay establishes feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

Chimeric peptide EP45 as a dual agonist at GLP-1 and NPY2R receptors

We report the design and target validation of chimeric peptide EP45, a novel 45 amino acid monomeric dual agonist peptide that contains amino acid sequence motifs present within the blood glucose-lowering agent exendin-4 (Ex-4) and the appetite-suppressing agent PYY(3–36). In a new high-throughput FRET assay that provides real-time kinetic information concerning levels of cAMP in living cells, EP45 recapitulates the action of Ex-4 to stimulate cAMP production via the glucagon-like peptide-1 receptor (GLP-1R), while also recapitulating the action of PYY(3–36) to inhibit cAMP production via the neuropeptide Y2 receptor (NPY2R). EP45 fails to activate glucagon or GIP receptors, whereas for cells that co-express NPY2R and adenosine A2B receptors, EP45 acts in an NPY2R-mediated manner to suppress stimulatory effects of adenosine on cAMP production. Collectively, such findings are remarkable in that they suggest a new strategy in which the co-existing metabolic disorders of type 2 diabetes and obesity will be treatable using a single peptide such as EP45 that lowers levels of blood glucose by virtue of its GLP-1R-mediated effect, while simultaneously suppressing appetite by virtue of its NPY2R-mediated effect.

Detection of Small-Molecule Modulators of Actin-Myosin Structure and Function using High-Throughput Time-Resolved FRET

We have used a time-resolved FRET assay to develop a system for detecting small-molecule modulators of actin-myosin structure and function. The fluorescent donor was fluorescein attached to actin C374, and the non-fluorescent acceptor was Dabcyl attached to the N-terminus of a peptide containing the 12 N-terminal amino acids of the A1 essential light chain (DNT). FRET was measured from the decrease in fluorescence lifetime, measured by time-resolved fluorescence in a novel fluorescence plate-reader, resolving structural distributions of the bound complex. The DNT binding site on actin overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-DNT complex as in the actin-myosin complex and (b) a significant decrease in actin-DNT FRET upon binding skeletal and cardiac myosin. A high-throughput FRET screen of a small-molecule library (NCC) showed that actin-DNT FRET is significantly affected by some compounds. All of these compounds inhibit actin-activated skeletal and cardiac myosin ATPase and affect the microsecond dynamics of actin, as detected by transient phosphorescence anisotropy. We concluded that the actin-DNT system allows detection of the effects of pharmacologically active compounds on actin structural states and consequently on functional properties of actomyosin. These results set the stage for a drug-discovery campaign, seeking therapies for cardiomyopathy and other disorders of involving actin-myosin dysfunction.

An in Vitro Sample Generation Pipeline for High-Throughput Single-Molecule FRET Based Screening of Proteins

An in Vitro Sample Generation Pipeline for High-Throughput Single-Molecule FRET Based Screening of Proteins

Toehold-Mediated Displacement of an Adenosine-Binding Aptamer from a DNA Duplex by its Ligand.

DNA is increasingly used to engineer dynamic nanoscale circuits, structures, and motors, many of which rely on DNA strand-displacement reactions. The use of functional DNA sequences (e.g., aptamers, which bind to a wide range of ligands) in these reactions would potentially confer responsiveness on such devices, and integrate DNA computation with highly varied molecular stimuli. By using high-throughput single-molecule FRET methods, we compared the kinetics of a putative aptamer-ligand and aptamer-complement strand-displacement reaction. We found that the ligands actively disrupted the DNA duplex in the presence of a DNA toehold in a similar manner to complementary DNA, with kinetic details specific to the aptamer structure, thus suggesting that the DNA strand-displacement concept can be extended to functional DNA-ligand systems.

A Two-Color Non-Muscle SERCA FRET Sensor for Diabetes Drug Discovery Using Fluorescence Lifetime Detection

We have developed intramolecular FRET sensors capable of detecting cytoplasmic headpiece movements of human SERCA (sarco/endo-plasmic reticulum calcium ATPase) in live-cell assays, including a non-muscle SERCA2b isoform to be used in drug discovery for treatment of diabetes. Two fluorescent proteins, clover (green) and mRuby2 (red) were directly fused to selected locations on human SERCA1a (skeletal muscle), 2a (cardiac muscle), and 2b (non muscle), based on a previously reported SERCA2a construct (Gruber et al., J. Biol. Screening, 2014), and expressed stably in HEK cells. We have used these cells in a novel fluorescence lifetime plate reader (FLT-PR) to screen small-molecule libraries, to discover modulators of SERCA structure and function. The present study focuses on SERCA2b, with the goal of obtaining small molecules that activate SERCA in non-muscle cells. Since recent reports indicate that SERCA overexpression in non-muscle cells can alleviate Type II diabetes, we seek small-molecule SERCA activators for the same purpose. The small-molecule modulators identified in the high-throughput FRET screen were examined for their ability to affect SERCA's function, through assays of ATPase and calcium pumping activities. In order to obtain functional data more directly related to Type II diabetes, we tested the compound's alleviation of endoplasmic reticulum stress in 3T3-L1 adipocytes, using an XF24 Extracellular Flux Analyzer to measure mitochondrial function after inducing ER stress with the inflammatory cytokine TNF-α. While this study is designed to find activators of SERCA2b for treatment of diabetes, constructs based on other SERCA isoforms show promise in targeted therapeutics for muscular dystrophy (SERCA1a) and heart failure (SERCA2a).

1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose inhibits angiogenesis via inhibition of capillary morphogenesis gene 2.

Capillary morphogenesis gene 2 (CMG2) is a transmembrane extracellular matrix binding protein that is also an anthrax toxin receptor. We have shown that high-affinity CMG2 binders can inhibit angiogenesis and tumor growth. We recently described a high-throughput FRET assay to identify CMG2 inhibitors. We now report the serendipitous discovery that PGG (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose) is a CMG2 inhibitor with antiangiogenic activity. PGG is a gallotannin produced by a variety of medicinal plants that exhibits a wide variety of antitumor and other activities. We find that PGG inhibits CMG2 with a submicromolar IC50 and it also inhibits the migration of human dermal microvascular endothelial cells at similar concentrations in vitro. Finally, oral or intraperitoneal administration of PGG inhibits angiogenesis in the mouse corneal micropocket assay in vivo. Together, these results suggest that a portion of the in vivo antitumor activity of PGG may be the result of antiangiogenic activity mediated by inhibition of CMG2.

Screening of drugs by FRET analysis identifies inhibitors of SARS-CoV 3CL protease

SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examined, hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concentrations ( K i = 4 μM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biological evaluation of various hexachlorophene analogues were described. These analogues gave optimal inhibitory activity against SARS-CoV 3CL protease with IC 50 values ranging from 7.6 to 84.5 μM. Optimization of hexachlorophene analogues was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.