A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins.

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

Phospholamban and sarcolipin prevent thermal inactivation of sarco(endo)plasmic reticulum Ca2+-ATPases.

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

Dietary docosahexaenoic acid supplementation reduces SERCA Ca2+ transport efficiency in rat skeletal muscle.

Docosahexaenoic acid (DHA) can reduce the efficiency and increase the energy consumption of Na(+)/K(+)-ATPase pump and mitochondrial electron transport chain by promoting Na(+) and H(+) membrane permeability, respectively. In skeletal muscle, the sarco(endo) plasmic reticulum Ca(2+)-ATPase (SERCA) pumps are major contributors to resting metabolic rate. Whether DHA can affect SERCA efficiency remains unknown. Here, we examined the hypothesis that dietary supplementation with DHA would reduce Ca(2+) transport efficiency of the SERCA pumps in skeletal muscle. Total lipids were extracted from enriched sarcoplasmic reticulum (SR) membranes that were isolated from red vastus lateralis skeletal muscles of rats that were either fed a standard chow diet supplemented with soybean oil or supplemented with DHA for 8 weeks. The fatty acid composition of total SR membrane lipids and the major phospholipid species were determined using electrospray ionization mass spectrometry (ESI-MS). After 8 weeks of DHA supplementation, total SR DHA content was significantly elevated (control, 4.1 ± 1.0% vs. DHA, 9.9 ± 1.7%; weight percent of total fatty acids) while total arachidonic acid was reduced (control, 13.5 ± 0.4% vs. DHA-fed, 9.4 ± 0.2). Similar changes in these fatty acids were observed in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, altogether indicating successful incorporation of DHA into the SR membranes post-diet. As hypothesized, DHA supplementation reduced SERCA Ca(2+) transport efficiency (control, 0.018 ± 0.0002 vs. DHA-fed, 0.014 ± 0.0009) possibly through enhanced SR Ca(2+) permeability (ionophore ratio: control, 2.8 ± 0.2 vs. DHA-fed, 2.2 ± 0.3). Collectively, our results suggest that DHA may promote skeletal muscle-based metabolism and thermogenesis through its influence on SERCA.

Cardio-protection by Ginkgo biloba extract 50 in rats with acute myocardial infarction is related to Na⁺-Ca²âº exchanger.

Ginkgo biloba has been used for medical purposes for centuries in traditional Chinese medicine. Ginkgo biloba extract 50 (GBE50) is a new standardized GBE product that matches the standardized German product as EGb761. This paper is aimed at studying the cardio-protection effects of GBE50 Salvia miltiorrhiza on myocardial function, area at risk, myocardial ultra-structure, and expression of calcium handling proteins in rat ischemic myocardium. Myocardium ischemia was induced by the left anterior descending (LAD) coronary artery occlusion and myocardial function was recorded by a transducer advanced into the left ventricle on a computer system. In vitro myocardial infarction was measured by 2,3,5-triphenyltetrazolium chloride (TTC) and Evans blue staining of heart sections. Morphological change was evaluated by electric microscopy and Western blotting was used for protein expression. Hemodynamic experiments in vivo showed that postischemic cardiac contractile function was reduced in ischemic rats. Salvia miltiorrhiza (7.5 g/kg/d×7) and Ginkgo biloba extract 50 (GBE50) (100 mg/kg/d×7) improved post-schemic cardiac diastolic dysfunction while not affecting the systolic function. In hearts of GBE50 group and Salvia miltiorrhiza (SM) group, the area at risk was significantly reduced and myocardial structure was better-preserved. Moreover, Na⁺-Ca²âº exchanger (NCX) expression increase and sarcoplasmic reticulum Ca²âº-ATPase 2 (SERCA2), LTCC, and ryanodine receptor 2 (RyR2) expression decreases were smaller than those in ischemia group. There was a significant difference between the GBE50 and ischemia group in NCX expression. GBE50 could improve recovery in contractile function and prevent myocardium from ischemia damage, which may be caused by attenuating the abnormal expression of NCX.

Testosterone regulates cardiac contractile activation by modulating SERCA but not NCX activity.

Alterations in intracellular Ca(2+) transients of cardiomyocytes in orchidectomized (ORX) rats could be a cause of cardiac dysfunction in the hypogonadal condition. To investigate the role of male sex hormones in intracellular Ca(2+) homeostasis during relaxation, Ca(2+)-handling activities by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and the Na(+)/Ca(2+) exchanger (NCX) were evaluated in the ventricular muscle of 10-wk-old ORX rats with and without testosterone supplementation (2.5 mg/kg testosterone propionate, 2 times/wk). ORX induced a 50% decrease in contraction force accompanied by a prolonged time to achieve 50% relaxation (T(50)) in isolated intact ventricular trabeculae, which was partially corrected by testosterone administration. Maximum active tension was also suppressed in ORX rats without changes in myofilament Ca(2+) sensitivity and passive stiffness of the heart. Using a sarcoplasmic reticulum-enriched membrane preparation, the maximum thapsigargin-sensitive SERCA activity of the ORX rat was 27% lower with an increased Ca(2+) sensitivity, which was prevented by testosterone treatment. However, neither changes in SERCA content nor its modulating components, sarcolipin and heat shock protein 20, were detected in the ORX rat, but there was a significant decrease in the phosphorylated Thr(17) form of phospholamban. Despite a lower level of NCX protein in the heart of ORX rats, prolonged T(50) disappeared after an incubation with thapsigargin (10 µM), implying a lack of effect of male sex hormone deficiency on NCX function. These findings indicate that male sex hormones can regulate cardiac relaxation by acting mainly through SERCA. However, a detailed mechanism of SERCA modulation under male sex hormone deficiency status remains to be explored.

Protective effect of oxymatrine on chronic rat heart failure.

Oxymatrine is one of the alkaloids extracted from the Chinese herb Sophora japonica (Sophora flavescens Ait.) with anti-inflammatory, immune reaction inhibiting, antiviral, and hepatocyte and antihepatic fibrosis protective activities. However, the effect of oxymatrine on heart failure is not yet known. In this study, the effect of oxymatrine on heart failure was investigated using a Sprague-Dawley rat model of chronic heart failure. Morphological findings showed that in the group treated with 50 and 100 mg/kg of oxymatrine; intermyofibrillar lysis disappeared, myofilaments were orderly, closely and evenly arranged; and mitochondria contained tightly packed cristae compared with the heart failure group. We investigated the cytosolic Ca(2+) transients and sarcoplasmic reticulum (SR) Ca(2+) content, and assessed the expression of ryanodine receptor (RyR2), SR-Ca(2+) ATPase (SERCA2a), and L-type Ca(2+) channel (dihydropyridine receptor, DHPR). We found that the cytosolic Ca(2+) transients were markedly increased in amplitude in the medium- (ΔF/F (0) = 26.22 ± 2.01) and high-dose groups (ΔF/F (0) = 29.49 ± 1.17) compared to the heart failure group (ΔF/F (0) = 12.12 ± 1.35, P < 0.01), with changes paralleled by a significant increase in the SR Ca(2+) content (medium-dose group: ΔF/F (0) = 32.20 ± 1.67, high-dose group: ΔF/F (0) = 32.57 ± 1.29, HF: ΔF/F (0) = 17.26 ± 1.05, P < 0.01). Moreover, we demonstrated that the expression of SERCA2a and cardiac DHPR was significantly increased in the medium- and high-dose group compared with the heart failure rats. These findings suggest that oxymatrine could improve heart failure by improving the cardiac function and that this amelioration is associated with upregulation of SERCA2a and DHPR.

In vitro effect of valepotriates isolated from Valeriana glechomifolia on rat P-type ATPases.

Valepotriates are iridoids found in variable amounts in Valerianaceae and might be among the bioactive compounds which confer anxiolytic properties to the Valeriana species. On the other hand, unspecific cytotoxicity has also been described. Presently, however, no particular molecular target has been defined for these compounds. Here we studied the effect of valtrate, acevaltrate, and 1- ß-acevaltrate isolated from Valeriana glechomifolia on the enzymatic activity of rat P-type ATPases. Valepotriates did not affect rat skeletal muscle sarco/endoplasmic reticulum Ca²âº-ATPase (SERCA) activity at the highest concentration used (100 µM). In contrast, the same concentration inhibited roughly half of the total H⁺/K⁺-ATPase activity from rat gastric epithelium (valtrate 54.6 ± 3.2 %, acevaltrate 60.7 ± 7.3 %, 1- ß-acevaltrate 50.2 ± 3.1 %; mean ± SEM, n = 3-5). Finally, these substances showed the highest inhibitory potency toward Na⁺/K⁺-ATPase, and the inhibition curves obtained provided a similar IC50 (in µM) for rat kidney α1 isoform (valtrate 21.2, acevaltrate 22.8, 1- ß-acevaltrate 24.4) and brain hemispheres α2/ α3 isoforms (valtrate 19.4, acevaltrate 42.3, 1- ß-acevaltrate 38.3). Our results suggest that P-type ATPases are differentially inhibited by valepotriates and that Na⁺/K⁺-ATPase might be one of their molecular targets in vivo.

The cardiac force-frequency relationship and frequency-dependent acceleration of relaxation are impaired in lipopolysaccharide-treated rats: is the phospholamban-SERCA axis a therapeutic target?

Sepsis-induced myocardial dysfunction has traditionally been thought of as principally affecting systolic heart function. One of the primary reasons for this concept is that systolic dysfunction is relatively easy to conceptualize, visualize, and measure. With the advent of preload-independent measurements for diastolic function, both measurement and conceptual difficulties are being resolved, and a new realm of evidence is beginning to emerge regarding the aberrations that are found during cardiac relaxation in sepsis. A recent article in Critical Care brings this issue into sharper focus.

Protective effect of antioxidants against sarcoplasmic reticulum (SR) oxidation by Fenton reaction, however without prevention of Ca-pump activity.

The Ca(2+)-ATPase of the sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle was oxidized by Fe2+/H2O2/ascorbic acid (AA), a system which generates HO(.) radicals according to the Fenton reaction: (Fe2(+)+H2O2-->HO(.)+OH(-)+Fe(3+)) under conditions similar to the pathological state of inflammation. Under these conditions, when hydroxyl-radicals and/or ferryl-radicals are generated, a 50% decrease of the SERCA activity was observed, a significant decrease of SH groups and an increase of protein carbonyl groups and lipid peroxidation were identified. Two new bands, time dependent in density, appeared in the SERCA protein electrophoresis after incubation with the Fenton system (at approximately 50 and 75kDa), probably due to structural changes as supported also by trypsin digestion. Immunoblotting of DNPH derivatized protein bound carbonyls detected a time dependent increase after incubation of SERCA with the Fenton system. Trolox and the pyridoindole stobadine (50microM) protected SR against oxidation induced via the Fenton system by preventing SH group oxidation and lipid peroxidation. Pycnogenol((R)) and EGb761 (40microg/ml) protected SERCA in addition against protein bound carbonyl formation. In spite of the antioxidant effects, trolox and stobadine were not able to prevent a decrease in the SERCA Ca(2+)-ATPase activity. Pycnogenol and EGb761 even enhanced the decrease of the Ca(2+)-ATPase activity induced by the Fenton system, probably by secondary oxidative reactions.

Cardioprotective effect of salvianolic acid B on large myocardial infarction mediated by reversing upregulation of leptin, endothelin pathways, and abnormal expression of SERCA2a, phospholamban in rats.

AIM: To study the cardioprotective effect of salvianolic acid B (Sal B) on cardiac dysfunction. We hypothesized that hyperleptinemia may correlate with abnormal expression of sarco/endoplasmic reticulum ATPase 2a (SERCA2a), phospholamban (PLB) and endothelin-reactive oxygen species (ET-ROS) pathways in rats with large myocardial infarction (MI). METHODS: Large MI was produced by coronary artery ligation for 4 weeks in rats. The rats were divided into four groups: sham, MI, MI+l-Sal B (50 mg/(kg d)), p.o. for 4 weeks), and MI+h-Sal B (100 mg/(kg d)), p.o. for 4 weeks). RESULTS: In MI rats, hemodynamic and echocardiographic abnormalities, cardiac remodeling, and histological changes with features of cardiac failure were associated with hyperleptinemia accompanied by oxidative stress and upregulated OB-Rb, ET pathway mRNA expression and downregulated SERCA2a and PLB mRNA and protein expressions in the myocardium. CONCLUSIONS: The studies demonstrated that an activated leptin pathway correlated with abnormal expression of SERCA2a, PLB and an activated ET-ROS system in the affected myocardium. Sal B exerts beneficial actions on cardiac function in rats with large MI, mainly suppressing upregulation of leptin and ET pathways and oxidative stress, and recovering the normal expressions of SERCA2a and PLB in myocardium.