An Open Drug Discovery Competition: Experimental Validation of Predictive Models in a Series of Novel Antimalarials.

The Open Source Malaria (OSM) consortium is developing compounds that kill the human malaria parasite, Plasmodium falciparum, by targeting PfATP4, an essential ion pump on the parasite surface. The structure of PfATP4 has not been determined. Here, we describe a public competition created to develop a predictive model for the identification of PfATP4 inhibitors, thereby reducing project costs associated with the synthesis of inactive compounds. Competition participants could see all entries as they were submitted. In the final round, featuring private sector entrants specializing in machine learning methods, the best-performing models were used to predict novel inhibitors, of which several were synthesized and evaluated against the parasite. Half possessed biological activity, with one featuring a motif that the human chemists familiar with this series would have dismissed as "ill-advised". Since all data and participant interactions remain in the public domain, this research project "lives" and may be improved by others.

Indole: The After Next Scaffold of Antiplasmodial Agents?

Malaria remains a global public health problem due to the uphill fight against the causative Plasmodium parasites that are relentless in developing resistance. Indole-based antiplasmodial compounds are endowed with multiple modes of action, of which inhibition of hemozoin formation is the major mechanism of action reported for compounds such as cryptolepine, flinderoles, and isosungucine. Indole-based compounds exert their potent activity against chloroquine-resistant Plasmodium strains by inhibiting hemozoin formation in a mode of action different from that of chloroquine or through a novel mechanism of action. For example, dysregulating the sodium and osmotic homeostasis of Plasmodium through inhibition of PfATP4 is the novel mechanism of cipargamin. The potential of developing multi-targeted compounds through molecular hybridization ensures the existence of indole-based compounds in the antimalarial pipeline.

Effects of Covalent Conjugates of Fullerene Derivatives with Xanthene Dyes on Activity of Ca2+-ATPase of the Sarcoplasmic Reticulum.

The effects of the newly synthesized covalent conjugates of water-soluble fullerene derivatives (WSFD) with xanthene dyes: polyanionic WSFD-fluorescein (1), polycationic WSFD-fluorescein (2), polyanionic WSFD-eosin (3), and polyanionic WSFD (4), polycationic WSFD (5), fluorescein (6) and eosin (7), on activity of the membrane-bound Ca2+-ATPase of the sarcoplasmic reticulum (SR Ca2+-ATPase) were studied. Compounds 1, 3, 4, 6, and 7 inhibit the hydrolytic function of the enzyme, the inhibition constants for these compounds are Ki=1.3×10-5 M (1), Ki=4.7×10-6 M (3), Ki=2.5×10-6 M (4), Ki=6.1×10-5 M (6), and Ki=5.8×10-6 M (7). The effects of compounds 3, 6, and 7 on the hydrolytic function of the enzyme is competitive; compounds 1 and 4 are noncompetitive. Polycationic WSFD fluorescein (2) and polycationic WSFD (5) do not affect ATP hydrolysis, but inhibit active Ca2+ transport in a concentration of 0.01 mM by 100±10 and 40±4%, respectively. Conjugates 1 and 3 completely inhibit the hydrolytic and transport functions of the enzyme in a concentration of 0.01 mM, and in a concentration of 0.001 mM inhibit active Ca2+ transport by 60±6 and 55±6% uncoupling the hydrolytic and transport functions of SR Ca2+-ATPases. The obtained results demonstrate a significant effect of the studied compounds on the active transmembrane transfer of Ca2+ and make it possible to predict the presence of antimetastatic and antiaggregatory activities of the studied compounds.

Neuroleptic Chlorpromazine Modulates Ca2+ Responses in Macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor antagonist neuroleptic chlorpromazine significantly inhibits glutoxim- and molixan-induced Ca2+ responses and Ca2+ responses induced by endoplasmic reticulum Са2+-ATPase inhibitors thapsigargin and cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in the signaling cascade induced by glutoxim or molixan and leading to intracellular Ca2+ concentration increase and in the regulation of store-dependent Ca2+ entry in macrophages.

Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p.

The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry. The enzyme complex exists in an autoinhibited form in the absence of an activator and is specifically activated by phosphatidylinositol-4-phosphate (PI4P), although the underlying mechanisms have been unclear. Here we report the cryo-EM structures of intact Drs2p-Cdc50p isolated from S. cerevisiae in apo form and in the PI4P-activated form at 2.8 Å and 3.3 Å resolution, respectively. The structures reveal that the Drs2p C-terminus lines a long groove in the cytosolic regulatory region to inhibit the flippase activity. PIP4 binding in a cytosol-proximal membrane region triggers a 90° rotation of a cytosolic helix switch that is located just upstream of the inhibitory C-terminal peptide. The rotation of the helix switch dislodges the C-terminus from the regulatory region, activating the flippase.

Thapsigargin, Inhibitor of Sarco-Endoplasmic Ca2+-ATPase, Effectively Suppresses the Expression of S100A4 Protein in Human Breast Cancer Cell Line.

Thapsigargin (SERCA ATPase inhibitor) inhibited the S100A4 metastatic marker expression in MDA-MB231 breast cancer cells. We found that S100A4 gene transcription is regulated by Ca2+ signaling pathways. We found that the synthesis of S100A4 mRNA and S100A4 protein in MDA-MB231 cells was effectively suppressed by thapsigargin at a concentration of 0.4-4 µM with retaining cell viability. We assume that the change in the gene transcription in response to disturbance of Ca2+ homeostasis is directly involved in the remodeling of Ca2+ signaling pathways.

The regulatory roles of calcium channels in tumors.

Calcium (Ca2+) and its relevant transmembrane and intracellular calcium channels were previously thought to be chiefly associated with the regulation of cardiovascular and neuronal systems. Nowadays, an increasing evidence shows those calcium channels are also responsible for tumorigenesis and progression. However, the general underlying mechanisms and the involving signaling transduction pathways still remain unclear. Therefore, in this mini-review, we are mainly focusing on the linkage between calcium channels and major characteristics of tumors such as multi-drug resistance (MDR), metastasis, apoptosis, proliferation, evasion of immune surveillance, and the alterations of tumor microenvironment. We will shed light on the possible therapeutic approaches to counteract tumors regarding the intervention of calcium channel.

Structure and autoregulation of a P4-ATPase lipid flippase.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.

Natural flavonoids inhibit the plasma membrane Ca2+-ATPase.

Research on flavonoids from plant sources has recently sparked increasing interest because of their beneficial health properties. Different studies have shown that flavonoids change the intracellular Ca2+ homeostasis linked to alterations in the function of mitochondria, Ca2+ channels and Ca2+ pumps. These findings hint at plasma membrane Ca2+-ATPase (PMCA) involvement, as it transports Ca2+ actively to the extracellular medium coupled to ATP hydrolysis, thus maintaining ion cellular homeostasis. The present study aims to investigate the effect of several natural flavonoids on PMCA both in isolated protein systems and in living cells, and to establish the relationship between flavonoid structure and inhibitory activity on PMCA. Our results show that natural flavonoids inhibited purified and membranous PMCA with different effectiveness: quercetin and gossypin were the most potent and their inhibition mechanisms seem to be different, as quercetin does not prevent ATP binding whereas gossypin does. Moreover, PMCA activity was inhibited in human embryonic kidney cells which transiently overexpress PMCA, suggesting that the effects observed on isolated systems could occur in a complex structure like a living cell. In conclusion, this work reveals a novel molecular mechanism through which flavonoids inhibit PMCA, which leads to Ca2+ homeostasis and signaling alterations in the cell.

Inhibition of Na+/K+- and Ca2+-ATPase activities by phosphotetradecavanadate.

Polyoxometalates (POMs) are promising inorganic inhibitors for P-type ATPases. The experimental models used to study the effects of POMs on these ATPases are usually in vitro models using vesicles from several membrane sources. Very recently, some polyoxotungstates, such as the Dawson anion [P2W18O62]6-, were shown to be potent P-type ATPase inhibitors; being active in vitro as well as in ex-vivo. In the present study we broaden the spectrum of highly active inhibitors of Na+/K+-ATPase from basal membrane of epithelial skin to the bi-capped Keggin-type anion phosphotetradecavanadate Cs5.6H3.4PV14O42 (PV14) and we confront the data with activity of other commonly encountered polyoxovanadates, decavanadate (V10) and monovanadate (V1). The X-ray crystal structure of PV14 was solved and contains two trans-bicapped α-Keggin anions HxPV14O42(9-x)-. The anion is built up from the classical Keggin structure [(PO4)@(V12O36)] capped by two [VO] units. PV14 (10 µM) exhibited higher ex-vivo inhibitory effect on Na+/K+-ATPase (78%) than was observed at the same concentrations of V10 (66%) or V1 (33%). Moreover, PV14 is also a potent in vitro inhibitor of the Ca2+-ATPase activity (IC50 5 µM) exhibiting stronger inhibition than the previously reported activities for V10 (15 µM) and V1 (80 µM). Putting it all together, when compared both P-typye ATPases it is suggested that PV14 exibited a high potential to act as an in vivo inhibitor of the Na+/K+-ATPase associated with chloride secretion.

Diverse Chemical Compounds Target Plasmodium falciparum Plasma Membrane Lipid Homeostasis.

Lipid homeostasis is essential to the maintenance of life. We previously reported that disruptions of the parasite Na+ homeostasis via inhibition of PfATP4 resulted in elevated cholesterol within the parasite plasma membrane as assessed by saponin sensitivity. A large number of compounds have been shown to target the parasite Na+ homeostasis. We screened 800 compounds from the Malaria and Pathogen Boxes to identify chemotypes that disrupted the parasite plasma membrane lipid homeostasis. Here, we show that the compounds disrupting parasite Na+ homeostasis also induced saponin sensitivity, an indication of parasite lipid homeostasis disruption. Remarkably, 13 compounds were identified that altered the plasma membrane lipid composition independently of the Na+ homeostasis disruption. Further studies suggest that these compounds target the Plasmodium falciparum Niemann-Pick type C1-related (PfNCR1) protein, which is hypothesized to be involved in maintaining plasma membrane lipid composition. PfNCR1, like PfATP4, appears to be targeted by multiple chemotypes with the potential for drug discovery.

E2P-like states of plasma membrane Ca2+­ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues.

The plasma membrane Ca2+­ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeFx), aluminium fluoride (AlFx) and magnesium fluoride (MgFx) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·Pi product states. Our results show that the phosphate analogues BeFx, AlFx and MgFx inhibit PMCA Ca2+­ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca2+­ATPase inhibition by AlFx and BeFx depended on Mg2+ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlFx and BeFx but not MgFx apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E → E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA. In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

BACKGROUND/AIMS: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. METHODS: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. RESULTS: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). CONCLUSION: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes.

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na+-dependent ATPase activity present in P. falciparum membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in P. falciparum in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na+ transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K+] or [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km for Na+ of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na+ efflux to increase in response to an increase in cytosolic [Na+]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

Design, synthesis and anti-malarial activities of synthetic analogs of biselyngbyolide B, a Ca2+ pump inhibitor from marine cyanobacteria.

Biselyngbyaside, an 18-membered macrolide glycoside from marine cyanobacteria, and its derivatives are known to be sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors. Recently, a SERCA orthologue of the malaria parasite, PfATP6, has attracted attention as a malarial drug target. To provide a novel drug lead, we designed new synthetic analogs of biselyngbyolide B, the aglycone of biselyngbyaside, based on the co-crystal structure of SERCA with biselyngbyolide B, and synthesized them using the established synthetic route for biselyngbyolide B. Their biological activities against malarial parasites were evaluated.

Calcium dynamics in tomato pollen tubes using the Yellow Cameleon 3.6 sensor.

KEY MESSAGE: In vitro tomato pollen tubes show a cytoplasmic calcium gradient that oscillates with the same period as growth. Pollen tube growth requires coordination between the tip-focused cytoplasmic calcium concentration ([Ca2+]cyt) gradient and the actin cytoskeleton. This [Ca2+]cyt gradient is necessary for exocytosis of small vesicles, which contributes to the delivery of new membrane and cell wall at the pollen tube tip. The mechanisms that generate and maintain this [Ca2+]cyt gradient are not completely understood. Here, we studied calcium dynamics in tomato (Solanum lycopersicum) pollen tubes using transgenic tomato plants expressing the Yellow Cameleon 3.6 gene under the pollen-specific promoter LAT52. We use tomato as an experimental model because tomato is a Solanaceous plant that is easy to transform, and has an excellent genomic database and genetic stock center, and unlike Arabidopsis, tomato pollen is a good system to do biochemistry. We found that tomato pollen tubes showed an oscillating tip-focused [Ca2+]cyt gradient with the same period as growth. Then, we used a pharmacological approach to disturb the intracellular Ca2+ homeostasis, evaluating how the [Ca2+]cyt gradient, pollen germination and in vitro pollen tube growth were affected. We found that cyclopiazonic acid (CPA), a drug that inhibits plant PIIA-type Ca2+-ATPases, increased [Ca2+]cyt in the subapical zone, leading to the disappearance of the Ca2+ oscillations and inhibition of pollen tube growth. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of Ca2+ released from the endoplasmic reticulum to the cytoplasm in animals cells, completely reduced [Ca2+]cyt at the tip of the tube, blocked the gradient and arrested pollen tube growth. Although both drugs have antagonistic effects on [Ca2+]cyt, both inhibited pollen tube growth triggering the disappearance of the [Ca2+]cyt gradient. When CPA and 2-APB were combined, their individual inhibitory effects on pollen tube growth were partially compensated. Finally, we found that GsMTx-4, a peptide from spider venom that blocks stretch-activated Ca2+ channels, inhibited tomato pollen germination and had a heterogeneous effect on pollen tube growth, suggesting that these channels are also involved in the maintenance of the [Ca2+]cyt gradient. All these results indicate that tomato pollen tube is an excellent model to study calcium dynamics.

Functional characterization of the Ca2+-ATPase SMA1 from Schistosoma mansoni.

Schistosoma mansoni is a parasite that causes bilharzia, a neglected tropical disease affecting hundreds of millions of people each year worldwide. In 2012, S. mansoni had been identified as the only invertebrate possessing two SERCA-type Ca2+-ATPases, SMA1 and SMA2. However, our analysis of recent genomic data shows that the presence of two SERCA pumps is rather frequent in parasitic flatworms. To understand the reasons of this redundancy in S. mansoni, we compared SMA1 and SMA2 at different levels. In terms of sequence and organization, the genes SMA1 and SMA2 are similar, suggesting that they might be the result of a duplication event. At the protein level, SMA1 and SMA2 only slightly differ in length and in the sequence of the nucleotide-binding domain. To get functional information on SMA1, we produced it in an active form in Saccharomyces cerevisiae, as previously done for SMA2. Using phosphorylation assays from ATP, we demonstrated that like SMA2, SMA1 bound calcium in a cooperative mode with an apparent affinity in the micromolar range. We also showed that SMA1 and SMA2 had close sensitivities to cyclopiazonic acid but different sensitivities to thapsigargin, two specific inhibitors of SERCA pumps. On the basis of transcriptomic data available in GeneDB, we hypothesize that SMA1 is a housekeeping Ca2+-ATPase, whereas SMA2 might be required in particular striated-like muscles like those present the tail of the cercariae, the infecting form of the parasite.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a).

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.

SC83288 is a clinical development candidate for the treatment of severe malaria.

Severe malaria is a life-threatening complication of an infection with the protozoan parasite Plasmodium falciparum, which requires immediate treatment. Safety and efficacy concerns with currently used drugs accentuate the need for new chemotherapeutic options against severe malaria. Here we describe a medicinal chemistry program starting from amicarbalide that led to two compounds with optimized pharmacological and antiparasitic properties. SC81458 and the clinical development candidate, SC83288, are fast-acting compounds that can cure a P. falciparum infection in a humanized NOD/SCID mouse model system. Detailed preclinical pharmacokinetic and toxicological studies reveal no observable drawbacks. Ultra-deep sequencing of resistant parasites identifies the sarco/endoplasmic reticulum Ca2+ transporting PfATP6 as a putative determinant of resistance to SC81458 and SC83288. Features, such as fast parasite killing, good safety margin, a potentially novel mode of action and a distinct chemotype support the clinical development of SC83288, as an intravenous application for the treatment of severe malaria.

Caffeoylquinic acids from antiplasmodial active extract of Xanthium cavanillesii fruits and their molecular modelling studies.

The antiplasmodial active extract of Xanthium cavanillesii contains 3,4-dicaffeoyl quinic acid (3,4-DCQA), 3,5-dicaffeoyl quinic acid (3,5-DCQA) and 1,3,5-tricaffeoyl quinic acid (1,3,5-TCQA). These results inspired us to investigate the interaction of these molecules with a promising validated target of Plasmodium, PfATP6 orthologue of mammalian Ca+2-ATPase. Models of this receptor were obtained through comparative modelling. Afterwards, molecular docking studies were used to identify possible interaction modes of these caffeoyl quinic derivatives on the binding site. The 1,3,5-TCQA had the best energy, but all of these had better energy than thapsigargin, a non-competitive inhibitor of the sarco/endoplasmatic reticulum Ca+2-ATPase (SERCA).