Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening.

We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.

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.

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA.

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA's distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA's ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells.

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

Fluorescence lifetime plate reader: resolution and precision meet high-throughput.

We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications.

Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.

We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

Dose range finding study for the efficacy of meloxicam administered prior to sodium urate-induced synovitis in cats.

OBJECTIVE: To determine the lowest efficacious dose of oral meloxicam for relieving pain in cats with a sodium urate (SU)-induced acute inflammatory synovitis. STUDY DESIGN: Randomized, blinded, controlled, and four-way crossover study. ANIMALS: Eight surgically neutered cats (four males, four females) paired according to sex. METHODS: Each pair of cats was treated with 0 (placebo), 0.025, 0.05, or 0.075 mg kg(-1) oral meloxicam once daily for 4 days prior to injection, into alternating stifles, of 1 mL of 20 mg mL(-1) SU crystals, beginning with the right stifle. Each cat received each of the four treatments, separated by at least 21 days. Analgesic efficacy was evaluated based on objective (e.g., pressure mat data total force, contact pressure, and contact area) and subjective (e.g., scores for Analgesia Scale [AS], Lameness Scale [LS], and Visual Analog Scale [VAS]) outcome measures for pain assessment. All outcome measures were recorded before and during 30 hours after SU injection. The pre-defined primary outcome measure was the area under the response-time curve (AUC(0-30) hours) of the total force of the injected limb. Data were analyzed by analysis of variance. A sequential test procedure was applied and the test sequence stopped in case of a nonsignificant result. RESULTS: Meloxicam at doses of 0.05 and 0.075 mg kg(-1) day(-1) PO was significantly different from placebo for the pre-defined primary outcome measure (i.e., AUC(0-30) hours of total force). All tested meloxicam doses were lower than placebo for the subjective outcome measures (i.e., AUC(0-30) hours of AS, LS, and VAS). CONCLUSIONS AND CLINICAL RELEVANCE: The lowest efficacious dose of meloxicam for relieving pain in cats with an SU-induced synovitis was 0.05 mg kg(-1) day(-1) PO according to the pre-defined primary outcome measure. However, lower doses may also be effective as seen in the subjective outcome measures.

Analgesic efficacy of preoperative administration of meloxicam or butorphanol in onychectomized cats.

OBJECTIVE: To determine analgesic efficacy and adverse effects of preemptive administration of meloxicam or butorphanol in cats undergoing onychectomy or onychectomy and neutering. DESIGN: Randomized controlled study. ANIMALS: 64 female and 74 male cats that were 4 to 192 months old and weighed 1.09 to 705 kg (2.4 to 15.5 lb). PROCEDURE: Cats received meloxicam (0.3 mg/kg [0.14 mg/lb], s.c.) or butorphanol (0.4 mg/kg [0.18 mg/lb], s.c.) 15 minutes after premedication and prior to anesthesia. A single blinded observer measured physiologic variables, assigned analgesia and lameness scores, and withdrew blood samples for each cat at baseline and throughout the 24 hours after surgery. Rescue analgesia (butorphanol, 0.4 mg/kg, i.v. or s.c.) or administration of acepromazine (0.025 to 0.05 mg/kg [0.011 to 0.023 mg/lb], i.v.) was allowed. RESULTS: Meloxicam-treated cats were less lame and had lower pain scores. Cortisol concentration was higher at extubation and lower at 1, 5, and 12 hours in the meloxicam-treated cats. Fewer meloxicam-treated cats required rescue analgesia at 3, 5, 12, and 24 hours after extubation. General impression scores were excellent or good in 75% of meloxicam-treated cats and 44% of butorphanol-treated cats. There was no treatment effect on buccal bleeding time; PCV and BUN concentration decreased in both groups, and glucose concentration decreased in meloxicam-treated cats. CONCLUSIONS AND CLINICAL RELEVANCE: Preoperative administration of meloxicam improved analgesia for 24 hours without clinically relevant adverse effects in cats that underwent onychectomy or onychectomy and neutering and provided safe, extended analgesia, compared with butorphanol.

Effects of meloxicam on severity of lameness and other clinical signs of osteoarthritis in dogs.

OBJECTIVE: To evaluate effects of meloxicam on severity of lameness and other clinical signs in dogs with osteoarthritis (OA). DESIGN: Randomized, controlled, multicenter clinical trial. ANIMALS: 217 client-owned dogs with clinical and radiographic signs of OA. PROCEDURE: Dogs were randomly assigned to be treated with meloxicam (n = 105; 0.2 mg/kg [0.09 mg/lb], SC, once on day 1, then 0.1 mg/kg [0.045 mg/lb], PO, q 24 h, for 13 days) or a placebo (n = 112). A general clinical score was assigned by investigators on days 1 (ie, prior to initiation of treatment), 8, and 15 on the basis of severity of lameness, extent of weight bearing, and severity of signs during palpation of the affected joint. Owners and investigators provided overall evaluations on days 8 and 15. RESULTS: Dogs treated with meloxicam had significantly greater improvements in general clinical scores, compared with baseline scores, on days 8 and 15 than did dogs treated with placebo. On days 8 and 15, percentages of dogs treated with meloxicam in which owners and investigators considered treatment to be successful were significantly higher than percentages of control dogs in which treatment was considered to be successful. No abnormalities in hematologic and serum biochemical test results were detected. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that compared with administration of a placebo, administration of meloxicam for 14 days significantly improved the clinical condition of dogs with OA without causing adverse effects.