We have developed a structure-based high-throughput screening (HTS) method, using time-resolved fluorescence resonance energy transfer (TR-FRET) that is sensitive to protein-protein interactions in living cells. The membrane protein complex between the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) and phospholamban (PLB), its Ca-dependent regulator, is a validated therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug. We co-expressed GFP-SERCA2a (donor) in the endoplasmic reticulum membrane of HEK293 cells with RFP-PLB (acceptor), and measured FRET using a fluorescence lifetime microplate reader. We screened a small-molecule library and identified 21 compounds (Hits) that changed FRET by >3SD. 10 of these Hits reproducibly alter SERCA2a-PLB structure and function. One compound increases SERCA2a calcium affinity in cardiac membranes but not in skeletal, suggesting that the compound is acting specifically on the SERCA2a-PLB complex, as needed for a drug to mitigate deficient calcium transport in heart failure. The excellent assay quality and correlation between structural and functional assays validate this method for large-scale HTS campaigns. This approach offers a powerful pathway to drug discovery for a wide range of protein-protein interaction targets that were previously considered “undruggable”.

Elevated cytoplasmic [Ca 2+ ] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca 2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [ 3 H]ryanodine binding to RyR1 at nanomolar Ca 2+. Both compounds also decrease RyR1 Ca 2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca 2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance. In striated muscle, contraction requires an intracellular Ca 2+-release event mediated by ryanodine receptors (RyR) that are embedded in the sarcoplasmic reticulum (SR) membrane. Dysregulation of skeletal (RyR1) and cardiac (RyR2) isoforms, via mutations or excess posttranslational modification, has been linked to severe muscle pathologies, including malignant hyperthermia (MH), central core disease, muscular dystrophy (MD), sarcopenia, catecholaminergic polymorphic ventricular tachycardia, heart failure, and more recently RyR2 has been recognized as a potentially significant contributor to diabetes and Alzheimer's disease 1-9. In most of these clinical indications, pathogenesis can be fueled by excess SR Ca 2+ "leak" via RyR under resting cellular conditions, which leads to toxic intracellular basal [Ca 2+ ] and insufficient SR Ca 2+ load. As a result, RyR is intensely studied as a therapeutic target. Indeed, the therapeutic potential of pharmaceutically targeting RyR1-mediated SR Ca 2+ leak in skeletal muscle has been shown in animal models of Duchenne MD, limb-girdle MD, and sarcopenia 5,10,11. The therapeutic potential of targeting RyR2-mediated SR Ca 2+ leak for treating heart failure and arrhythmia is also very well documented 12-16. Additionally, targeting RyR2 (which is abundant in the brain 17,18) may have therapeutic potential for treating neurodegenerative diseases. To introduce a systematic and efficient approach for identifying novel small-molecule chemical scaffolds with potential to mitigate RyR1 dysfunction, we developed and implemented a high-throughput screening (HTS) assay that uses fluorescence lifetime (FLT) detection of FRET 19. This assay was designed to identify compounds that bind to the RyR1 channel complex to allosterically correct its pathologically leaky state (without affecting normal channel function) 19. This FRET-based method is based on monitoring RyR binding of fluorescent...

pathway heterogeneity, we develop a Monte Carlo computational approach for investigating the issue using discrete-state (reaction network) models. The calculations yield families of models, some exhibiting multiple pathways, which are consistent with a fitness goal (e.g., driven transport against a concentration gradient). Many of the models would be difficult to find by intuition alone in the complex state-spaces of interest. Our approach can also address the related issue of investigating what functions are possible for molecular machines. As an example, we study transporters in a complex environment where it is necessary to discriminate against decoy ligands; computations reveal models with non-integer stoichiometry that exploit excess free energy from an ion leak to perform driven ''proofreading'' -i.e., enhanced selectivity. Gram negative bacteria are reliant on TonB dependent transporters for the movement of scarce compounds across their outer membranes. These proteins consist of an outer b-barrel surrounding an internal hatch domain. The substrates, including iron siderophores and cobalamin, are large and would necessitate significant conformational rearrangement of the hatch domain to facilitate their movement. In vitro work on the E. coli cobalamin transporter, BtuB, using the pulsed EPR technique double electron-electron resonance (DEER) in our lab has thus far failed to produce evidence of such conformational changes within the hatch domain. Recently, we have focused on the development of techniques to study the protein in intact systems, including whole cells and isolated outer membranes. While investigating hatch-barrel mutant pairs in these systems, we have seen evidence for conformational changes in the whole cells that are induced by the presence of substrate, and that are absent or greatly suppressed in the isolated membrane alone. These results may reveal elements of the transport process, as well as highlighting the importance of studying proteins in their native environments. TonB dependent transporters (TBDTs) are a family of outer membrane proteins (OMPs) in Gram-negative bacteria that facilitate the active transport of nutrients across the outer membrane (OM). The energy for active transport is derived from inner membrane (IM) proton motive force (pmf) through a reversible coupling to IM protein TonB. Our current understanding of the molecular function of TBDTs is mainly based on high-resolution structural and biophysical characterizations in detergent purified and reconstituted lipid vesicles. Unfortunately, TBDT has never been reconstituted and structural measurements have never been made on an active transport system. Recent work has shown that it is possible to spin label the OMP, BtuB in Gram-negative bacteria, thereby enabling the study of TBDT systems using EPR spectroscopy. 1 In our present work, we altered the labeling strategy to allow attachment of methanethiosulfonate spin label in cells that are optimized for their metabolic activity. In these preparations we find that specific...

By applying well designed oscillating electrical pulse to a single frog skeletal muscle with voltage-clamp and double vaseline gap techniques, we obtained pump current on both positive and negative half cycle. The frequency of the oscillating pulses remains at 50 Hz which is comparable with the physiological turnover rate of the Na/K pump. Different magnitudes of voltage (from 30mV to 65mV with a 5mV step) were applied to the muscle. The results showed that the pump current as well as the charge mediated by pump moved across the plasma cell membrane were voltage dependent and reached a saturation status when the pulse was greater than 55 mV. In this situation, the total amount of pump-mediated charge remained roughly the same even with the changing magnitude of the activation pulses. The charge on the positive half cycle and negative half cycle which represent the Na extrusion and K intrusion respectively, has a ratio less than but close to 3:2. After adding ouabain, the currents were recorded each 5 minutes. The results showed that the total pump-mediated charge decreased monotonically and reached minimum which was around 0 after 20 minutes. This indicated that this oscillating electrical pulse induced current was highly sensitive to and can be totally eliminated by ouabain, a specific Na/K pump blocker.