Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

Structural Dynamics of Protein Interactions Using Site-Directed Spin Labeling of Cysteines to Measure Distances and Rotational Dynamics with EPR Spectroscopy.

Here we review applications of site-directed spin labeling (SDSL) with engineered cysteines in proteins, to study the structural dynamics of muscle and non-muscle proteins, using and developing the electron paramagnetic resonance (EPR) spectroscopic techniques of dipolar EPR, double electron electron resonance (DEER), saturation transfer EPR (STEPR), and orientation measured by EPR. The SDSL technology pioneered by Wayne Hubbell and collaborators has greatly expanded the use of EPR, including the measurement of distances between spin labels covalently attached to proteins and peptides. The Thomas lab and collaborators have applied these techniques to elucidate dynamic interactions in the myosin-actin complex, myosin-binding protein C, calmodulin, ryanodine receptor, phospholamban, utrophin, dystrophin, ß-III-spectrin, and Aurora kinase. The ability to design and engineer cysteines in proteins for site-directed covalent labeling has enabled the use of these powerful EPR techniques to measure distances, while showing that they are complementary with optical spectroscopy measurements.

Regulation of cardiac calcium signaling by newly identified calcium pump modulators.

In cardiomyocytes, the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a central component of intracellular Ca2+ regulation. Several heart diseases, including heart failure, are associated with reduced myocardial contraction due to SERCA2a downregulation. Therefore, the need for developing new drugs that could improve SERCA2a function is high. We have recently identified SERCA2a modulators (Compounds 6 and 8) from our screening campaigns and confirmed activation of biochemical SERCA2a ATPase activity and Ca2+ uptake activity. In this study, confocal microscopy and in-cell Ca2+ imaging were used to characterize the effects of these SERCA2a activators on Ca2+ regulation in mouse ventricular myocytes and endoplasmic reticulum (ER) Ca2+ uptake in a HEK293 cell expressing human SERCA2a. Analysis of cytosolic Ca2+ dynamics in cardiomyocytes revealed that both Compounds (6 and 8) increase the action potential-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load. While Compound 6 induced a negligible effect on Ca2+ transients invoked by the L-type Ca2+ channel (LTCC) current, Compound 8 increased Ca2+ transients during LTCC activation, suggesting an off-target protein interaction of Compound 8. Analysis of ER Ca2+ transport by human SERCA2a in HEK cells showed that only Compound 6 increased both ER Ca2+ uptake and ER Ca2+ load significantly, whereas Compound 8 had no effect on SERCA2a Ca2+ transport. This study revealed that Compound 6 exhibits promising characteristics that can improve intracellular Ca2+ dynamics by selectively enhancing SERCA2a Ca2+ uptake.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here, we used fluorescence lifetime (FLT)-measurements of Förster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State.

Ca2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide (DPc10) binding promotes leaky RyR2 in cardiomyocytes and reports on that endogenous state. Conversely, calmodulin (CaM) binding inhibits RyR2 leak and low CaM affinity is diagnostic of leaky RyR2. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. We used FRET to clarify the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with the analysis of fluorescence-lifetime-detected FRET, indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, with this effect being larger in micromolar versus nanomolar Ca2+. Moreover, A-DPc10/RyR2 binding is cooperative in a CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by the analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA's function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein.

There is a critical need for small molecules capable of rescuing pathophysiological phenotypes induced by alpha-synuclein (aSyn) misfolding and oligomerization. Building upon our previous aSyn cellular fluorescence lifetime (FLT)-Förster resonance energy transfer (FRET) biosensors, we have developed an inducible cell model incorporating the red-shifted mCyRFP1/mMaroon1 (OFP/MFP) FRET pair. This new aSyn FRET biosensor improves the signal-to-noise ratio, reduces nonspecific background FRET, and results in a 4-fold increase (transient transfection) and 2-fold increase (stable, inducible cell lines) in FRET signal relative to our previous GFP/RFP aSyn biosensors. The inducible system institutes greater temporal control and scalability, allowing for fine-tuning of biosensor expression and minimizes cellular cytotoxicity due to overexpression of aSyn. Using these inducible aSyn-OFP/MFP biosensors, we screened the Selleck library of 2684 commercially available, FDA-approved compounds and identified proanthocyanidins and casanthranol as novel hits. Secondary assays validated the ability of these compounds to modulate aSyn FLT-FRET. Functional assays probing cellular cytotoxicity and aSyn fibrillization demonstrated their capability to inhibit seeded aSyn fibrillization. Proanthocyanidins completely rescued aSyn fibril-induced cellular toxicity with EC50 of 200 nM and casanthranol supported a 85.5% rescue with a projected EC50 of 34.2 µM. Furthermore, proanthocyanidins provide a valuable tool compound to validate our aSyn biosensor performance in future high-throughput screening campaigns of industrial-scale (million-compound) chemical libraries.

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State.

Ca 2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide, (DPc10) detects leaky RyR2 in cardiomyocytes. Conversely, calmodulin (CaM) inhibits RyR2 leak. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. Here we used FRET to understand the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca 2+ , DPc10 decreased both FRET max (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca 2+ , DPc10 decreased FRET max without affecting CaM/RyR2 binding affinity. This correlates with analysis of fluorescence-lifetime-detected FRET indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca 2+ and lengthens D-FKBP/A-CaM distances independent of [Ca 2+ ]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, this effect being larger in micromolar vs. nanomolar Ca 2+ . Moreover, A-DPc10/RyR2 binding is cooperative in CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

Cardiomyopathy-associated variants alter the structure and function of the α-actinin-2 actin-binding domain.

Hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM) are characterized by thickening, thinning, or stiffening, respectively, of the ventricular myocardium, resulting in diastolic or systolic dysfunction that can lead to heart failure and sudden cardiac death. Recently, variants in the ACTN2 gene, encoding the protein α-actinin-2, have been reported in HCM, DCM, and RCM patients. However, functional data supporting the pathogenicity of these variants is limited, and potential mechanisms by which these variants cause disease are largely unexplored. Currently, NIH ClinVar lists 34 ACTN2 missense variants, identified in cardiomyopathy patients, which we predict are likely to disrupt actin binding, based on their localization to specific substructures in the α-actinin-2 actin binding domain (ABD). We investigated the molecular consequences of three ABD localized, HCM-associated variants: A119T, M228T and T247 M. Using circular dichroism, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all three mutations are destabilizing, suggesting a structural disruption. Importantly, A119T decreased actin binding, and M228T and T247M cause increased actin binding. We suggest that altered actin binding underlies pathogenesis for cardiomyopathy mutations localizing to the ABD of α-actinin-2.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin-myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces binding of cMyBP-C to actin or myosin, is often decreased in heart failure (HF) patients, and is cardioprotective in model systems for HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL), and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethyl rhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding and/or structural changes in the protein complex. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fskC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish feasibility for discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Indirubin Inhibits TRAIL-Induced Activation of Death Receptor 5 in Jurkat Cells.

Death receptor 5 (DR5) is an apoptosis-inducing membrane receptor that mediates cell death in several life-threatening conditions. There is a crucial need for the discovery of DR5 antagonists for the therapeutic intervention of conditions in which the overactivation of DR5 underlies the pathophysiology. DR5 activation mediates cell death in non-alcoholic fatty liver disease (NAFLD) and neurodegenerative processes including amyloid-beta (Aß) accumulation, spinal cord injury (SCI), and brain ischemia. In the current work, we used fluorescence resonance energy transfer (FRET) to monitor the conformational dynamics of DR5 that mediate death signaling. We used a time-resolved FRET screening platform to screen the Selleck library of 2863 U.S. Food and Drug Administration (FDA)-approved compounds. The high-throughput screen (HTS) identified 13 compounds that modulated the FRET between DR5 monomers beyond 5 median absolute deviations (MADs) from the DMSO controls. Of these 13 compounds, indirubin was identified to specifically inhibit tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 activity without modulating DR5 surface expression or TRAIL binding. Indirubin inhibited Fas-associated death domain (FADD) oligomerization and increased cellular FLICE-inhibitory protein (c-FLIP) expression; both are molecular mechanisms involved in inhibiting the DR5 signaling cascade. This study has elucidated previously unknown properties of indirubin that make it a promising candidate for therapeutic investigation of diseases in which overactivation of DR5 underlies pathology.

FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca 2+ -ATPase and Ca 2+ -transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca 2+ -ATPase and Ca 2+ -transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Early-phase drug discovery of ß-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.

ß-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein ß-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.

Nimesulide, a COX-2 inhibitor, sensitizes pancreatic cancer cells to TRAIL-induced apoptosis by promoting DR5 clustering †.

Nimesulide is a nonsteroidal anti-inflammatory drug and a COX-2 inhibitor with antitumor and antiproliferative activities that induces apoptosis in oral, esophagus, breast, and pancreatic cancer cells. Despite being removed from the market due to hepatotoxicity, nimesulide is still an important research tool being used to develop new anticancer drugs. Multiple studies have been done to modify the nimesulide skeleton to develop more potent anticancer agents and related compounds are promising scaffolds for future development. As such, establishing a mechanism of action for nimesulide remains an important part of realizing its potential. Here, we show that nimesulide enhances TRAIL-induced apoptosis in resistant pancreatic cancer cells by promoting clustering of DR5 in the plasma membrane. In this way, nimesulide acts like a related compound, DuP-697, which sensitizes TRAIL-resistant colon cancer cells in a similar manner. Our approach applies a time-resolved FRET-based biosensor that monitors DR5 clustering and conformational states in the plasma membrane. We show that this tool can be used for future high-throughput screens to identify novel, nontoxic small molecule scaffolds to overcome TRAIL resistance in cancer cells.

Interaction of DWORF with SERCA and PLB as determined by EPR spectroscopy.

Insufficient sarco/endoplasmic reticulum calcium ATPase (SERCA) activity significantly contributes to heart failure, which is a leading cause of death worldwide. A characteristic pathology of cardiac disease is the slow and incomplete Ca2+ removal from the myocyte cytoplasm in diastole, which is primarily driven by SERCA, the integral transmembrane Ca2+ pump. Phospholamban (PLB) allosterically inhibits SERCA by reducing its apparent Ca2+ affinity. Recently, the 34-codon novel dwarf open reading frame (DWORF) micropeptide has been identified as a muscle-specific SERCA effector, capable of reversing the inhibitory effects of PLB and independently activating SERCA in the absence of PLB. However, the structural basis for these functions has not yet been determined in a system of defined molecular components. We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interactions of DWORF, co-reconstituted in proteoliposomes with SERCA and spin-labeled PLB. We analyzed the change of PLB rotational mobility in response to varying DWORF concentration, to quantify competitive binding of DWORF and PLB. We determined that DWORF competes with PLB for binding to SERCA at low [Ca2+], although the measured affinity of DWORF for SERCA is an order of magnitude weaker than that of PLB for SERCA, indicating cooperativity. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to obtain new information needed to refine the molecular model for regulation of SERCA activity, as needed for development of novel therapeutic remedies against cardiac pathologies.

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping.

The type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a central role in the intracellular Ca2+ homeostasis of cardiac myocytes, pumping Ca2+ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca2+ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca2+-ATPase and Ca2+ transport activities in cardiac SR vesicles, and on Ca2+ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca2+-ATPase and Ca2+ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca2+ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca2+ load by enhancing SERCA2a-mediated Ca2+ transport. Analysis of cytosolic Ca2+ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca2+ transients and SR Ca2+ load, with negligible effects on L-type Ca2+ channels and Na+/Ca2+ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca2+ transients and SR Ca2+ load, but it decreased the propensity toward Ca2+ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [3H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca2+ and a small increase in µM Ca2+. Accordingly, compound 1 slightly decreased Ca2+ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca2+ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca2+ uptake, as found in failing hearts.

A Large-Scale High-Throughput Screen for Modulators of SERCA Activity.

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ion pump that transports Ca2+ from the cytosol into the endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells. It is critically important in muscle, facilitating relaxation and enabling subsequent contraction. Increasing SERCA expression or specific activity can alleviate muscle dysfunction, most notably in the heart, and we seek to develop small-molecule drug candidates that activate SERCA. Therefore, we adapted an NADH-coupled assay, measuring Ca-dependent ATPase activity of SERCA, to high-throughput screening (HTS) format, and screened a 46,000-compound library of diverse chemical scaffolds. This HTS platform yielded numerous hits that reproducibly alter SERCA Ca-ATPase activity, with few false positives. The top 19 activating hits were further tested for effects on both Ca-ATPase and Ca2+ transport, in both cardiac and skeletal SR. Nearly all hits increased Ca2+ uptake in both cardiac and skeletal SR, with some showing isoform specificity. Furthermore, dual analysis of both activities identified compounds with a range of effects on Ca2+-uptake and ATPase, which fit into distinct classifications. Further study will be needed to identify which classifications are best suited for therapeutic use. These results reinforce the need for robust secondary assays and criteria for selection of lead compounds, before undergoing HTS on a larger scale.