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Stefan Paula

Publications and source records attributed to Stefan Paula.

7 recordsLinked to original sources
PubMed2005

Molecular modeling of cardiac glycoside binding by the human sequence monoclonal antibody 1B3.

The amino acid sequences of the heavy- and light-chain variable regions of the high-affinity human sequence antidigoxin monoclonal antibody 1B3 (mAb 1B3) were determined, and a structural model for the mAb's variable region was developed by homology modeling techniques. The structural model provided the basis for computationally docking digoxin and eight related cardiac glycosides into the putative binding site of mAb 1B3. Analysis of the consensus binding mode obtained for digoxin showed that the cardenolide moiety of digoxin is deeply embedded in a predominantly hydrophobic, narrow cavity, whereas the terminal, gamma-carbohydrate group is solvent-exposed. The docking results indicated that the primary driving forces for digoxin binding by mAb 1B3 are hydrophobic interactions with the digoxin steroid ring system and hydrogen bonds with the digitoxose groups. The binding model accounts for the experimentally observed variations in mAb 1B3 binding affinity for various structural analogs of digoxin used previously to develop a 3D structure-activity relationship model of drug binding (Farr CD, Tabet MR, Ball WJ Jr, Fishwild DM, Wang X, Nair AC, Welsh WJ. Three-dimensional quantitative structure-activity relationship analysis of ligand binding to human sequence antidigoxin monoclonal antibodies using comparative molecular field analysis. J Med Chem 2002;45:3257-3270). In particular, the hydrogen bond pattern is consistent with the unique sensitivity of mAb 1B3's binding affinity to the number of sugar residues present in a cardiac glycoside. The hydrophobic environment about the steroid moiety of digoxin is compatible with the mAb's reduced affinity for ligands that possess hydrophilic hydroxyl and acetyl group modifications in this region. The model also indicated that most of the amino acid residues in contact with the ligand reside in or about the three complementarity determining regions (CDRs) of the heavy chain and the third CDR of the light chain. A comparison of the 1B3 binding model with the crystal structures of two murine antidigoxin mAbs revealed similar binding patterns used by the three mAbs, such as a high frequency of occurrence of aromatic, hydrophobic residues in the CDRs and a dominant role of the heavy chain CDR3 in antigen binding.

Amino Acid Sequence↗
PubMed2005

Interactions between cardiac glycosides and sodium/potassium-ATPase: three-dimensional structure-activity relationship models for ligand binding to the E2-Pi form of the enzyme versus activity inhibition.

Sodium/potassium-ATPase (Na/K-ATPase) is a transmembrane enzyme that utilizes energy gained from ATP hydrolysis to transport sodium and potassium ions across cell membranes in opposite directions against their chemical and electrical gradients. Its transport activity is effectively inhibited by cardiac glycosides, which bind to the extracellular side of the enzyme and are of significant therapeutic value in the treatment of congestive heart failure. To determine the extent to which high-affinity binding of cardiac glycosides correlates with their potency in inhibiting pump activity, we determined experimentally both the binding affinities and inhibitory potencies of a series of 37 cardiac glycosides using radioligand binding and ATPase activity assays. The observed variations in key structural elements of these compounds correlating with binding and inhibition were analyzed by comparative molecular similarity index analysis (CoMSIA), which allowed a molecular level characterization and comparison of drug-Na/K-ATPase interactions that are important for ligand binding and activity inhibition. In agreement with our earlier comparative molecular field analysis studies [Farr, C. D., et al. (2002) Biochemistry 41, 1137-1148], the CoMSIA models predicted favorable inhibitor interactions primarily at the alpha-sugar and lactone ring moieties of the cardiac glycosides. Unfavorable interactions were located about the gamma-sugar group and at several positions about the steroid ring system. Whereas for most compounds a correlation between binding affinity and inhibitory potency was found, some notable exceptions were identified. Substitution of the five-membered lactone of cardenolides with the six-membered lactone of bufadienolides caused binding affinity to decline but inhibitory potency to increase. Furthermore, while the removal of ouabain's rhamnose moiety had little effect on inhibitory potency, it caused a dramatic decline in ligand binding affinity.

Algorithms↗
PubMed2005

Elucidation of the Na+, K+-ATPase digitalis binding site.

Despite controversy over their use and the potential for toxic side effects, cardiac glycosides have remained an important clinical component for the treatment for congestive heart failure (CHF) and supraventricular arrhythmias since the effects of Digitalis purpurea were first described in 1785. While there is a wealth of information available with regard to the effects of these drugs on their pharmacological receptor, the Na(+), K(+)-ATPase, the exact molecular mechanism of digitalis binding and inhibition of the enzyme has remained elusive. In particular, the absence of structural knowledge about Na(+), K(+)-ATPase has thwarted the development of improved therapeutic agents with larger therapeutic indices via rational drug design approaches. Here, we propose a binding mode for digoxin and several analogues to the Na(+), K(+)-ATPase. A 3D-structural model of the extracellular loop regions of the catalytic alpha1-subunit of the digitalis-sensitive sheep Na(+), K(+)-ATPase was constructed from the crystal structure of an E(1)Ca(2+) conformation of the SERCA1a and a consensus orientation for digitalis binding was inferred from the in silico docking of a series of steroid-based cardiotonic compounds. Analyses of species-specific enzyme affinities for ouabain were also used to validate the model and, for the first time, propose a detailed model of the digitalis binding site.

Amino Acid Sequence↗
PubMed2004

Molecular determinants of thapsigargin binding by SERCA Ca2+-ATPase: a computational docking study.

Thapsigargin (TG) is a potent and commonly used inhibitor of the ion transport activity of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). Based on the recently published crystal structures of rabbit muscle SERCA1a in the Ca2+/E1 (E1) and TG/E2 (E2) conformations, we performed computational docking studies to characterize the molecular interactions that govern binding of TG and TG-analogs by the enzyme. Using the program GOLD (genetic optimization for ligand docking) in combination with the scoring function ChemScore, TG was docked into the binding site of the E1 and E2 conformations of SERCA1a. The docking results revealed a consensus ligand-binding mode consistent with the crystal structure and showed that hydrophobic interactions are the primary driving force of TG binding by SERCA. Moreover, it was shown that the conformational changes accompanying the E2 to E1 transition in the enzyme likely displace TG from its favored orientation in the binding site, thereby substantially reducing its binding affinity. This finding illustrates on the molecular level how TG may exert its inhibitory effect in binding tightly to the E2 form and preventing it from converting into its E1 form, a requirement for catalytic function. We also docked 9 TG analogs into the E2 conformation of the enzyme. Eight of the analogs adopted a binding mode very similar to that of TG, whereas one compound preferred a different orientation in the binding site. Analysis of the predicted binding affinities showed a good correlation with the experimentally observed inhibitory potencies of the analogs. Docking was also performed with several modeled mutants of SERCA1a, whose phenylalanine residue in position 256 (Phe256) had been modified. The experimentally observed declines in TG sensitivity in most of the Phe256 mutants was qualitatively accounted for and appears, at least in part, be due to a slightly altered TG-binding mode.

Algorithms↗
PubMed2004

Three-dimensional quantitative structure-activity relationship modeling of cocaine binding by a novel human monoclonal antibody.

Human monoclonal antibodies (mAbs) designed for immunotherapy have a high potential for avoiding the complications that may result from human immune system responses to the introduction of nonhuman mAbs into patients. This study presents a characterization of cocaine/antibody interactions that determine the binding properties of the novel human sequence mAb 2E2 using three-dimensional quantitative structure-activity relationship (3D-QSAR) methodology. We have experimentally determined the binding affinities of mAb 2E2 for cocaine and 38 cocaine analogues. The K(d) of mAb 2E2 for cocaine was 4 nM, indicating a high affinity. Also, mAb 2E2 displayed good cocaine specificity, as reflected in its 10-, 1500-, and 25000-fold lower binding affinities for the three physiologically relevant cocaine metabolites benzoylecgonine, ecgonine methyl ester, and ecgonine, respectively. 3D-QSAR models of cocaine binding were developed by comparative molecular similarity index analysis (CoMSIA). A model of high statistical quality was generated showing that cocaine binds to mAb 2E2 in a sterically restricted binding site that leaves the methyl group attached to the ring nitrogen of cocaine solvent-exposed. The methyl ester group of cocaine appears to engage in attractive van der Waals interactions with mAb 2E2, whereas the phenyl group contributes to the binding primarily via hydrophobic interactions. The model further indicated that an increase in partial positive charge near the nitrogen proton and methyl ester carbonyl group enhances binding affinity and that the ester oxygen likely forms an intermolecular hydrogen bond with mAb 2E2. Overall, the cocaine binding properties of mAb 2E2 support its clinical potential for development as a treatment of cocaine overdose and addiction.

Animals↗
PubMed2003

Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis.

Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabolites by [3H]-radioligand binding assays. mAb 3P1A6 (K(d) = 0.22 nM) displayed a 50-fold higher affinity for cocaine than mAb MM0240PA (K(d) = 11 nM) and also had a greater specificity for cocaine. For the systematic exploration of both antibodies' binding specificities, we used a set of approximately 35 cocaine analogues as structural probes by determining their relative binding affinities (RBAs) using an enzyme-linked immunosorbent competition assay. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models on the basis of comparative molecular field analysis (CoMFA) techniques correlated the binding data with structural features of the ligands. The analysis indicated that despite the mAbs' differing specificities for cocaine, the relative contributions of the steric (approximately 80%) and electrostatic (approximately 20%) field interactions to ligand-binding were similar. Generated three-dimensional CoMFA contour plots then located the specific regions about cocaine where the ligand/receptor interactions occurred. While the overall binding patterns of the two mAbs had many features in common, distinct differences were observed about the phenyl ring and the methylester group of cocaine. Furthermore, using previously published data, a 3D-QSAR model was developed for cocaine binding to the dopamine reuptake transporter (DAT) that was compared to the mAb models. Although the relative steric and electrostatic field contributions were similar to those of the mAbs, the DAT cocaine-binding site showed a preference for negatively charged ligands. Besides establishing molecular level insight into the interactions that govern cocaine binding specificity by biopolymers, the three-dimensional images obtained reflect the properties of the mAbs binding pockets and provide the initial information needed for the possible design of novel antibodies with properties optimized for immunotherapy.

Animals↗