Druglike Molecules Binding to Large Membrane Proteins: Absolute Binding Free Energy Computation.

In this research, we employed the alchemical double-decoupling method alongside restraining potentials, coupled with the FEPMD method, to ascertain the standard binding free energy of a drug-like molecule termed BHQ and three analogous compounds engineered with progressive addition of bulky para-alkyl groups binding to SERCA (Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum). Integral transmembrane proteins represent crucial drug targets in numerous therapeutic interventions, presenting computational challenges due to their considerable system sizes. Our approach integrated the generalized born potential method and the spherical solvent boundary potential method, allowing us to explicitly focus on the active binding site while treating the remainder of the system implicitly. We evaluated contributions to the standard binding free energy from distinct interaction potentials: electrostatic, repulsive, dispersive, and restraining potentials, computed separately. The resulting absolute binding free energy of BHQ (11.63 kcal/mol) closely aligns with experimental measurements (10.56 kcal/mol). Notably, an accurate estimation of the absolute binding free energy was achieved for the simplest analog, created with the addition of a single para-methyl group. However, the analog with two para-methyl groups exhibited the highest binding free energy, which disagreed with experimental results. Determining the binding free energy of the BHQ analog engineered with three para-methyl groups presented challenges in convergence and resulted in the lowest free energy among the three.

The molecular determinants of calcium ATPase inhibition by curcuminoids.

The natural product curcumin and some of its analogs are known inhibitors of the transmembrane enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). Despite their widespread use, the curcuminoids' binding site in SERCA and their relevant interactions with the enzyme remain elusive. This lack of knowledge has prevented the development of curcuminoids into valuable experimental tools or into agents of therapeutic value. We used the crystal structures of SERCA in its E1 conformation in conjunction with computational tools such as docking and surface screens to determine the most likely curcumin binding site, along with key enzyme/inhibitor interactions. Additionally, we determined the inhibitory potencies and binding affinities for a small set of curcumin analogs. The predicted curcumin binding site is a narrow cleft in the transmembrane section of SERCA, close to the transmembrane/cytosol interface. In addition to pronounced complementarity in shape and hydrophobicity profiles between curcumin and the binding pocket, several hydrogen bonds were observed that were spread over the entire curcumin scaffold, involving residues on several transmembrane helices. Docking-predicted interactions were compatible with experimental observations for inhibitory potencies and binding affinities. Based on these findings, we propose an inhibition mechanism that assumes that the presence of a curcuminoid in the binding site arrests the catalytic cycle of SERCA by preventing it from converting from the E1 to the E2 conformation. This blockage of conformational change is accomplished by a combination of steric hinderance and hydrogen-bond-based cross-linking of transmembrane helices that require flexibility throughout the catalytic cycle.

Modelling energy-harvesting processes in primitive cells: Proton transport across bilayers driven by the oxidation of sulfite.

A frequently debated topic related to the origin of life centers around the question of how complex forms of life on today's Earth may have evolved over time from simpler predecessors. For example, the question of how proton concentration gradients across cellular membranes developed in ancestral protocells remains unanswered. This process, which is indispensable for the generation of chemical energy in modern organisms, is driven by energy derived from redox processes in the respiratory chain. Since it is highly unlikely that the complex machinery of the respiratory chain was available on early Earth, we provide an example of how proton gradients can be established in less complex systems. Utilizing liposomes as models of primitive cells, we were able to generate proton gradients of about two pH units across the liposome bilayers using redox reactions as the driving force. Electrons were transferred from sodium sulfite present on the outside of the liposomes to ferricyanide, which was trapped on the inside. A lipid-soluble phenazine derivative served as a shuttle that transferred both electrons and protons across the lipid bilayer. Because sulfite would have been an abundant reduced solute available to the earliest cells, we propose that it may have been a primary source of redox energy for primitive chemiosmotic energy transduction.

Development and interpretation of a QSAR model for in vitro breast cancer (MCF-7) cytotoxicity of 2-phenylacrylonitriles.

The Arylhydrocarbon Receptor (AhR), a member of the Per-ARNT-SIM transcription factor family, has been as a potential new target to treat breast cancer sufferers. A series of 2-phenylacrylonitriles targeting AhR has been developed that have shown promising and selective activity against cancerous cell lines while sparing normal non-cancerous cells. A quantitative structure-activity relationship (QSAR) modeling approach was pursued in order to generate a predictive model for cytotoxicity to support ongoing synthetic activities and provide important structure-activity information for new structure design. Recent work conducted by us has identified a number of compounds that exhibited false positive cytotoxicity values in the standard MTT assay. This work describes a good quality model that not only predicts the activity of compounds in the MCF-7 breast cancer cell line, but was also able to identify structures that subsequently gave false positive values in the MTT assay by identifying compounds with aberrant biological behavior. This work not only allows the design of future breast cancer cytotoxic activity in vitro, but allows the avoidance of the synthesis of those compounds anticipated to result in anomalous cytotoxic behavior, greatly enhancing the design of such compounds.

Modelling and Phenotypic Screening of NAP-6 and 10-Cl-BBQ, AhR Ligands Displaying Selective Breast Cancer Cytotoxicity in Vitro.

To exploit the interaction of the aryl hydrocarbon receptor (AhR) pathway in developing breast-cancer-specific cytotoxic compounds, we examined the breast cancer selectivity and the docking pose of the AhR ligands (Z)-2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (NAP-6; 5) and 10-chloro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (10-Cl-BBQ; 6). While the breast cancer selectivity of 5 in vitro is known, we discuss the SAR around this lead and, by using phenotypic cell-line screening and the MTT assay, show for the first time that 6 also presents with breast cancer selectivity, notably in the triple-negative (TN) receptor breast cancer cell line MDA-MB-468, the ER+ breast cancer cell lines T47D, ZR-75-1 and the HER2+ breast cancer cell line SKBR3 (GI50 values of 0.098, 0.97, 0.13 and 0.21 µM, respectively). Indeed, 6 is 55 times more potent in MDA-MB-468 cells than normal MCF10A breast cells (GI50 of 0.098 vs 5.4 µM) and more than 130 times more potent than in cell lines derived from pancreas, brain and prostate (GI50 of 0.098 vs 10-13 µM). Molecular docking poses of 5 and 6 together with analogue synthesis and phenotypic screening show the importance of the naphthalene moiety, and an ortho-disposed substituent on the N-phenyl moiety for biological activity.

Conservation of Cdc14 phosphatase specificity in plant fungal pathogens: implications for antifungal development.

Cdc14 protein phosphatases play an important role in plant infection by several fungal pathogens. This and other properties of Cdc14 enzymes make them an intriguing target for development of new antifungal crop treatments. Active site architecture and substrate specificity of Cdc14 from the model fungus Saccharomyces cerevisiae (ScCdc14) are well-defined and unique among characterized phosphatases. Cdc14 appears absent from some model plants. However, the extent of conservation of Cdc14 sequence, structure, and specificity in fungal plant pathogens is unknown. We addressed this by performing a comprehensive phylogenetic analysis of the Cdc14 family and comparing the conservation of active site structure and specificity among a sampling of plant pathogen Cdc14 homologs. We show that Cdc14 was lost in the common ancestor of angiosperm plants but is ubiquitous in ascomycete and basidiomycete fungi. The unique substrate specificity of ScCdc14 was invariant in homologs from eight diverse species of dikarya, suggesting it is conserved across the lineage. A synthetic substrate mimetic inhibited diverse fungal Cdc14 homologs with similar low µM Ki values, but had little effect on related phosphatases. Our results justify future exploration of Cdc14 as a broad spectrum antifungal target for plant protection.

Heck- and Suzuki-coupling approaches to novel hydroquinone inhibitors of calcium ATPase.

In this study, we explored Heck- and Suzuki-coupling methodology to modify the template 2,5-di-tert-butylhydroquinone (BHQ, 2), an inhibitor of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). We found that by utilizing Suzuki coupling, we could successfully attach a six-carbon tether to BHQ that terminated in a leucine moiety to obtain target 14. Similar to related compounds based on the structure of the natural product thapsigargin, 14 displayed inhibitory potency against SERCA activity. This makes 14 a suitable candidate for the future attachment of a deactivating peptide to convey specificity for prostate cancer cells.

Synthesis and characterization of CAPE derivatives as xanthine oxidase inhibitors with radical scavenging properties.

Inhibitors of the enzyme xanthine oxidase (XO) with radical scavenging properties hold promise as novel agents against reperfusion injuries after ischemic events. By suppressing the formation of damaging reactive oxygen species (ROS) by XO or scavenging ROS from other sources, these compounds may prevent a buildup of ROS in the aftermath of a heart attack or stroke. To combine these two properties in a single molecule, we synthesized and characterized the non-purine XO inhibitor caffeic acid phenethylester (CAPE) and 19 derivatives using a convenient microwave-assisted Knoevenagel condensation protocol. Varying systematically the number and positions of the hydroxyl groups at the two phenyl rings, we derived structure-activity relationships based on experimentally determined XO inhibition data. Molecular docking suggested that critical enzyme/inhibitor interactions involved π-π interactions between the phenolic inhibitor ring and Tyr914, hydrogen bonds between inhibitor hydroxyl groups and Glu802, and hydrophobic interactions between the CAPE phenyl ring and non-polar residues located at the entrance of the binding site. To effectively scavenge the stable radical DPPH, two hydroxyl groups in 1,2- or 1,4-position at the phenyl ring were required. Among all compounds tested, E-phenyl 3-(3,4-dihydroxyphenyl)acrylate, a CAPE analog without the ethyl tether, showed the most promising properties.

Dichlorophenylacrylonitriles as AhR Ligands That Display Selective Breast Cancer Cytotoxicity in vitro.

Knoevenagel condensation of 3,4-dichloro- and 2,6-dichlorophenylacetonitriles gave a library of dichlorophenylacrylonitriles. Our leads (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile (5) and (Z)-2-(3,4-dichlorophenyl)-3-(4-nitrophenyl)acrylonitrile (6) displayed 0.56±0.03 and 0.127±0.04 µm growth inhibition (GI50 ) and 260-fold selectivity for the MCF-7 breast cancer cell line. A 2,6-dichlorophenyl moiety saw a 10-fold decrease in potency; additional nitrogen moieties (-NO2 ) enhanced activity (Z)-2-(2,6-dichloro-3-nitrophenyl)-3-(2-nitrophenyl)acrylonitrile (26) and (Z)-2-(2,6-dichloro-3-nitrophenyl)-3-(3-nitrophenyl)acrylonitrile (27), with the corresponding -NH2 analogues (Z)-2-(3-amino-2,6-dichlorophenyl)-3-(2-aminophenyl)acrylonitrile (29) and (Z)-2-(3-amino-2,6-dichlorophenyl)-3-(3-aminophenyl)acrylonitrile (30) being more potent. Despite this, both 29 (2.8±0.03 µm) and 30 (2.8±0.03 µm) were found to be 10-fold less cytotoxic than 6. A bromine moiety effected a 3-fold enhancement in solubility with (Z)-3-(5-bromo-1H-pyrrol-2-yl)-2-(3,4-dichlorophenyl)acrylonitrile 18 relative to 5 at 211 µg mL-1 . Modeling-guided synthesis saw the introduction of 4-aminophenyl substituents (Z)-3-(4-aminophenyl)-2-(3,4-dichlorophenyl)acrylonitrile (35) and (Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide (38), with respective GI50 values of 0.030±0.014 and 0.034±0.01 µm. Other analogues such as 35 and 36 were found to have sub-micromolar potency against our panel of cancer cell lines (HT29, colon; U87 and SJ-G2, glioblastoma; A2780, ovarian; H460, lung; A431, skin; Du145, prostate; BE2-C, neuroblastoma; MIA, pancreas; and SMA, murine glioblastoma), except compound 38 against the U87 cell line. A more extensive evaluation of 38 ((Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide) in a panel of drug-resistant breast carcinoma cell lines showed 10-206 nm potency against MDAMB468, T47D, ZR-75-1, SKBR3, and BT474. Molecular Operating Environment docking scores showed a good correlation between predicted binding efficiencies and observed MCF-7 cytotoxicity. This supports the use of this model in the development of breast-cancer-specific drugs.

Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and radical scavengers.

In this study, we evaluated the abilities of a series of chalcones to inhibit the activity of the enzyme xanthine oxidase (XO) and to scavenge radicals. 20 mono- and polyhydroxylated chalcone derivatives were synthesized by Claisen-Schmidt condensation reactions and then tested for inhibitory potency against XO, a known generator of reactive oxygen species (ROS). In parallel, the ability of the synthesized chalcones to scavenge a stable radical was determined. Structure-activity relationship analysis in conjunction with molecular docking indicated that the most active XO inhibitors carried a minimum of three hydroxyl groups. Moreover, the most effective radical scavengers had two neighboring hydroxyl groups on at least one of the two phenyl rings. Since it has been proposed previously that XO inhibition and radical scavenging could be useful properties for reduction of ROS-levels in tissue, we determined the chalcones' effects to rescue neurons subjected to ROS-induced stress created by the addition of ß-amyloid peptide. Best protection was provided by chalcones that combined good inhibitory potency with high radical scavenging ability in a single molecule, an observation that points to a potential therapeutic value of this compound class.

Novel phenolic inhibitors of the sarco/endoplasmic reticulum calcium ATPase: identification and characterization by quantitative structure-activity relationship modeling and virtual screening.

Inhibitors of the sarco/endoplasmic reticulum calcium ATPase (SERCA) are valuable research tools and hold promise as a new generation of anti-prostate cancer agents. Based on previously determined potencies of phenolic SERCA inhibitors, we created quantitative structure-activity relationship (QSAR) models using three independent development strategies. The obtained QSAR models facilitated virtual screens of several commercial compound collections for novel inhibitors. Sixteen compounds were subsequently evaluated in SERCA activity inhibition assays and 11 showed detectable potencies in the micro- to millimolar range. The experimental results were then incorporated into a comprehensive master QSAR model, whose physical interpretation by partial least squares analysis revealed that properly positioned substituents at the central phenyl ring capable of forming hydrogen bonds and of undergoing hydrophobic interactions were prerequisites for effective SERCA inhibition. The established SAR was in good agreement with findings from previous structural studies, even though it was obtained independently using standard QSAR methodologies.

Investigation of fluorinated and bifunctionalized 3-phenylchroman-4-one (isoflavanone) aromatase inhibitors.

Fluorinated isoflavanones and bifunctionalized isoflavanones were synthesized through a one-step gold(I)-catalyzed annulation reaction. These compounds were evaluated for their in vitro inhibitory activities against aromatase in a fluorescence-based enzymatic assay. Selected compounds were tested for their anti-proliferative effects on human breast cancer cell line MCF-7. Compounds 6-methoxy-3-(pyridin-3-yl)chroman-4-one (3c) and 6-fluoro-3-(pyridin-3-yl)chroman-4-one (3e) were identified as the most potent aromatase inhibitors with IC50 values of 2.5 µM and 0.8 µM. Therefore, these compounds have great potential for the development of pharmaceutical agents against breast cancer.

Structural requirements for inhibitory effects of bisphenols on the activity of the sarco/endoplasmic reticulum calcium ATPase.

Bisphenols (BPs) are a class of small organic compounds with widespread industrial applications. Previous studies have identified several BPs that interfere with the activity of the ion-translocating enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). In order to define the molecular determinants of BP-mediated SERCA inhibition, we conducted enzyme activity assays with rabbit SERCA to determine the inhibitory potencies of 27 commercially available BPs, which were the basis for structure-activity relationships. The most potent BPs inhibited SERCA at low micromolar concentrations and carried at their two phenyl rings multiple non-polar substituents, such as small alkyl groups or halides. Furthermore, the presence of methyl groups or a cyclohexyl group at the central carbon atom connecting the two phenyl moieties correlated with good potencies. For a characterization and visualization of enzyme/inhibitor interactions, molecular docking was performed, which suggested that hydrogen bonding with Asp254 and hydrophobic interactions were the major driving forces for BP binding to SERCA. Calcium imaging studies with a selection of BPs showed that these inhibitors were able to increase intracellular calcium levels in living human cells, a behavior consistent with that of a SERCA inhibitor.

Development of a new class of aromatase inhibitors: design, synthesis and inhibitory activity of 3-phenylchroman-4-one (isoflavanone) derivatives.

Aromatase (CYP19) catalyzes the aromatization reaction of androgen substrates to estrogens, the last and rate-limiting step in estrogen biosynthesis. Inhibition of aromatase is a new and promising approach to treat hormone-dependent breast cancer. We present here the design and development of isoflavanone derivatives as potential aromatase inhibitors. Structural modifications were performed on the A and B rings of isoflavanones via microwave-assisted, gold-catalyzed annulation reactions of hydroxyaldehydes and alkynes. The in vitro aromatase inhibition of these compounds was determined by fluorescence-based assays utilizing recombinant human aromatase (baculovirus/insect cell-expressed). The compounds 3-(4-phenoxyphenyl)chroman-4-one (1h), 6-methoxy-3-phenylchroman-4-one (2a) and 3-(pyridin-3-yl)chroman-4-one (3b) exhibited potent inhibitory effects against aromatase with IC(50) values of 2.4 µM, 0.26 µM and 5.8 µM, respectively. Docking simulations were employed to investigate crucial enzyme/inhibitor interactions such as hydrophobic interactions, hydrogen bonding and heme iron coordination. This report provides useful information on aromatase inhibition and serves as a starting point for the development of new flavonoid aromatase inhibitors.

Discovery of novel SERCA inhibitors by virtual screening of a large compound library.

Two screening protocols based on recursive partitioning and computational ligand docking methodologies, respectively, were employed for virtual screens of a compound library with 345,000 entries for novel inhibitors of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA), a potential target for cancer chemotherapy. A total of 72 compounds that were predicted to be potential inhibitors of SERCA were tested in bioassays and 17 displayed inhibitory potencies at concentrations below 100 µM. The majority of these inhibitors were composed of two phenyl rings tethered to each other by a short link of one to three atoms. Putative interactions between SERCA and the inhibitors were identified by inspection of docking-predicted poses and some of the structural features required for effective SERCA inhibition were determined by analysis of the classification pattern employed by the recursive partitioning models.

Comparison of current docking tools for the simulation of inhibitor binding by the transmembrane domain of the sarco/endoplasmic reticulum calcium ATPase.

Inhibitors of the transmembrane protein sarco/endoplasmic reticulum calcium ATPase (SERCA) are invaluable tools for the study of the enzyme's physiological functions and they have been recognized as a promising new class of anticancer agents. For the discovery of novel enzyme inhibitors, small molecule docking for virtual screens of large compound libraries has become increasingly important. Since the performance of various docking routines varies considerably, depending on the target and the chemical nature of the ligand, we critically evaluated the performance of four frequently used programs - GOLD, AutoDock, Surflex-Dock, and FRED - for the docking of SERCA inhibitors based on the structures of thapsigargin, di-tert-butylhydroquinone, and cyclopiazonic acid. Evaluation criteria were docking accuracy using crystal structures as references, docking reproducibility, and correlation between docking scores and known bioactivities. The best overall results were obtained by GOLD and FRED. Docking runs with conformationally flexible binding sites produced no significant improvement of the results.

A molecular model for cocaine binding by the immunotherapeutic human/mouse chimeric monoclonal antibody 2E2.

Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (gamma1 heavy chain)/murine (lambda light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2's ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a homology model for the 3-D structure of its Fv fragment. Subsequent computational docking studies revealed the intermolecular interactions potentially responsible for mAb 2E2's cocaine binding properties. The driving force of cocaine binding was identified as a combination of hydrophobic interactions and a single hydrogen bond between a light chain tyrosine residue and a carbonyl oxygen atom of cocaine. The model also allowed for an in silico evaluation of single/double residue mutations in the heavy and light chain variable regions that might further enhance mAb 2E2's cocaine binding properties.

Design, synthesis, and biological evaluation of hydroquinone derivatives as novel inhibitors of the sarco/endoplasmic reticulum calcium ATPase.

Analogues of the compound 2,5-di-tert-butylhydroquinone (BHQ) are capable of inhibiting the enzyme sarco/endoplasmic reticulum ATPase (SERCA) in the low micromolar and submicromolar concentration ranges. Not only are SERCA inhibitors valuable research tools, but they also have potential medicinal value as agents against prostate cancer. This study describes the synthesis of 13 compounds representing several classes of BHQ analogues, such as hydroquinones with a single aromatic substituent, symmetrically and unsymmetrically disubstituted hydroquinones, and hydroquinones with omega-amino acid tethers attached to their hydroxyl groups. Structure-activity relationships were established by measuring the inhibitory potencies of all synthesized compounds in bioassays. The assays were complemented by computational ligand docking for an analysis of the relevant ligand/receptor interactions.

Structure-based virtual screening for novel inhibitors of the sarco/endoplasmic reticulum calcium ATPase and their experimental evaluation.

A public compound library with 260,000 compounds was screened virtually by computational docking for novel inhibitors of the transmembrane enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). Docking was performed with the program GOLD in conjunction with a high resolution X-ray crystal structure of SERCA. Compounds that were predicted to be active were tested in bioassays. Nineteen novel compounds were discovered that were capable of inhibiting the ATP hydrolysis activity of SERCA at concentrations below 50 microM. Crucial enzyme/inhibitor interactions were identified by analyzing the docking-predicted binding poses of active compounds. Like other SERCA inhibitors, the newly discovered compounds are of considerable medicinal interest because of their potential for cancer chemotherapy.

Molecular determinants of sarco/endoplasmic reticulum calcium ATPase inhibition by hydroquinone-based compounds.

The ion transport activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA) is specifically and potently inhibited by the small molecule 2,5-di-tert-butylhydroquinone (BHQ). In this study, we investigated the relative importance of the nature and position of BHQ's four substituents for enzyme inhibition by employing a combination of experimental and computational techniques. The inhibitory potencies of 21 commercially available or synthesized BHQ derivatives were determined in ATPase activity assays, and 11 compounds were found to be active. Maximum inhibitory potency was observed in compounds with two para hydroxyl groups, whereas BHQ analogues with only one hydroxyl group were still active, albeit with a reduced potency. The results also demonstrated that two alkyl groups were an absolute requirement for activity, with the most potent compounds having 2,5-substituents with four or five carbon atoms at each position. Using the program GOLD in conjunction with the ChemScore scoring function, the structures of the BHQ analogues were docked into the crystal structure of SERCA mimicking the enzyme's E(2) conformation. Analysis of the docking results indicated that inhibitor binding to SERCA was primarily mediated by a hydrogen bond between a hydroxyl group and Asp-59 and by hydrophobic interactions involving the bulky inhibitor alkyl groups. Attempts to dock BHQ into crystal structures corresponding to the E(1) conformation of the enzyme failed, because the conformational changes accompanying the E(2)/E(1) transition severely restricted the size of the binding site, suggesting that BHQ stabilizes the enzyme in its E(2) form. The potential role of Glu309 in enzyme inhibition is discussed in the context of the computational results. The docking scores correlated reasonably well with the measured inhibitory potencies and allowed the distinction between active and inactive compounds, which is a key requirement for future virtual screening of large compound databases for novel SERCA inhibitors.