William J. Ball

Analysis of the Na,K-ATPase α- and β-subunit expression profiles of bladder cancer using tissue microarrays

The purpose of this study was to determine the clinical significance of Na,K‐ATPase α‐ and β‐subunit expression in a histopathologically well‐characterized group of patients representing a wide spectrum of tumor grades and disease stages with transitional cell carcinomas (TCC).

A Chimeric Human/Murine Anticocaine Monoclonal Antibody Inhibits the Distribution of Cocaine to the Brain in Mice

The predominantly human sequence, high-affinity anticocaine monoclonal antibody (mAb) 2E2 was cleared slowly from mouse blood by a first-order process with an elimination t1/2 of 8.1 days. Infused 2E2 also produced a dramatic dose-dependent increase in plasma cocaine concentrations and a concomitant decrease in the brain cocaine concentrations produced by an i.v. injection of cocaine HCl (0.56 mg/kg). At the highest dose of 2E2 tested (3:1, mAb/drug), cocaine was not detectable in the brain. Pharmacokinetic studies showed that the normal disappearance of cocaine from plasma was described by a two-compartment pharmacokinetic model with distribution t1/2α and terminal elimination t1/2β values of 1.9 and 26.1 min, respectively. In the presence of an equimolar dose of mAb 2E2, there was a 26-fold increase in the area under the plasma cocaine concentration-time curve (AUC) relative to the AUC in the absence of 2E2. Consequently, 2E2 decreased the volume of distribution of cocaine from 6.0 to 0.20 l/kg, which approximated that of 2E2 (0.28 l/kg). However, cocaine was still rapidly cleared from plasma, and its elimination was now described by a single-compartment model with an elimination t1/2 of 17 min. Importantly, 2E2 also produced a 4.5-fold (78%) decrease in the cocaine AUC in the brain. Therefore, the effect of 2E2 on plasma and brain cocaine concentrations was predominantly caused by a change in the distribution of cocaine with negligible effects on its rate of clearance. These data support the concept of immunotherapy for drug abuse.

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. Proteins 2004.

Effect of quinidine on the digoxin receptor in vitro.

To investigate the basis for a clinically important digitalis-quinidine interaction that is characterized by increases in serums digoxin concentrations when quinidine is administered to digoxin-treated patients, we have studied in vitro the interaction of quinidine with the digoxin receptor. Evidence has been obtained that quinidine is capable of decreasing the affinity for digoxin of cardiac glycoside receptor sites on purified Na,K-ATPase and on intact human erythrocyte membranes. As others have shown, quinidine is capable of inhibiting Na,K-ATPase activity, and evidence has been obtained in the current study that, while quinidine can reduce the affinity of the enzyme for digoxin, it is also capable of acting together with digoxin in inhibiting enzyme activity to a degree greater than the inhibitory effect of digoxin alone. The concentrations of digoxin and quinidine used in this study were considerably greater than their therapeutic serum concentrations. Nevertheless, these observations are consistent with the hypothesis that the increases in serum digoxin concentrations and the decreases in volumes of digoxin distribution observed clinically when quinidine is administered to digoxin-treated patients may reflect, at least in part, a decrease in the affinity of tissue receptors for digoxin. The possibility must also be considered that enhanced cardiac effects of digoxin may occur clinically as the result of an augmentation, by quinidine, of digoxin effects, which more than compensates for the modest reduction in digoxin binding.

STRUCTURE, FUNCTION AND REGULATION OF Na-K-ATPase

Abstract 1. 1. The Na-K-ATPase consists of a large chain (α) and a small chain (β) and possibly a proteolipid chain (γ), of molecular weights 100,000, 45,000 and about 13,000 respectively. 2. 2. Various fluorescent-labelled probes when incorporated into purified lamb kidney Na-K-ATPase, reveal important conformational changes that appear to be modified by cations, including calcium. 3. 3. The regulation ofouabain binding to the Na-K-ATPase involves interaction of both Na+ and K+ at an “external” site. 4. 4. Vanadate may be involved in regulation of the sodium pump, by interaction with an “internal” site. This produces modification of an “external” K+ site. Both ATPase activity and ouabain binding are affected by this trace metal. 5. 5. A search is ongoing for endogenous factor(s) that specifically interact with the digitalis receptor and the Na-K-ATPase.

A Recombinant Humanized Anti-Cocaine Monoclonal Antibody Inhibits the Distribution of Cocaine to the Brain in Rats

The monoclonal antibody (mAb), h2E2, is a humanized version of the chimeric human/murine anti-cocaine mAb 2E2. The recombinant h2E2 protein was produced in vitro from a transfected mammalian cell line and retained high affinity (4 nM Kd) and specificity for cocaine over its inactive metabolites benzoylecgonine (BE) and ecgonine methyl ester. In rats, pharmacokinetic studies of h2E2 (120 mg/kg i.v.) showed a long terminal elimination half-life of 9.0 days and a low volume of distribution at steady state (Vdss) of 0.3 l/kg. Pretreatment with h2E2 produced a dramatic 8.8-fold increase in the area under the plasma cocaine concentration-time curve (AUC) and in brain a concomitant decrease of 68% of cocaine’s AUC following an i.v. injection of an equimolar cocaine dose. Sequestration of cocaine in plasma by h2E2, shown via reduction of cocaine’s Vdss, indicates potential clinical efficacy. Although the binding of cocaine to h2E2 in plasma should inhibit distribution and metabolism, the elimination of cocaine remained multicompartmental and was still rapidly eliminated from plasma despite the presence of h2E2. BE was the major cocaine metabolite, and brain BE concentrations were sixfold higher than in plasma, indicating that cocaine is normally metabolized in the brain. In the presence of h2E2, brain BE concentrations were decreased and plasma BE was increased, consistent with the observed h2E2-induced changes in cocaine disposition. The inhibition of cocaine distribution to the brain confirms the humanized mAb, h2E2, as a lead candidate for development as an immunotherapy for cocaine abuse.

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 mAbs 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, γ‐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 1B3s 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 mAbs 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. Proteins 2005.

Predicting the clinical efficacy and potential adverse effects of a humanized anticocaine monoclonal antibody.

The effects of a humanized monoclonal antibody (mAb) having high affinity and specificity for cocaine in animal models are reviewed. The mAb reduced the concentration of cocaine in the brain of mice after intravenous injection of cocaine. In addition, the mAb increased the concentration of cocaine required to reinstate cocaine self-administration. These effects may predict clinical efficacy of a passive immunotherapy for reducing the probability of cocaine-induced relapse. However, in the presence of the mAb, once cocaine self-administration was reinstated, the consumption rate of cocaine was increased. This effect is hypothesized to result from a pharmacokinetic/pharmacodynamic interaction. A humanized mAb should minimize adverse events related to the immunogenicity of the mAb protein, and the specificity for cocaine should avoid adverse events related to interactions with physiologically relevant endogenous proteins.

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 2E2s 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 2E2s 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 2E2s cocaine binding properties.

Anti-cocaine Monoclonal Antibodies

The use of an anti-cocaine monoclonal antibody (mAb) is similar to the concept of cocaine vaccines, discussed elsewhere in this book. Although vaccines have many advantages as an immunotherapy for cocaine abuse, the use of anti-cocaine mAbs addresses the limitations of cocaine vaccines: the polyclonal response that produces between-patient variability of affinities of the antibodies raised, the amount of antibodies raised, and the delay in the production of antibodies. Directly injecting therapeutically relevant doses of antibodies with known high affinity and selectivity avoids the between-patient variability in response to active immunization. Anti-cocaine mAbs could be used as a stand-alone treatment or as a supplement to patients treated with cocaine vaccines who do not produce adequate levels of high-affinity antibodies. Additionally, the antibodies are active immediately upon injection, whereas vaccines require time to elicit an immune response. Therefore, mAbs are better suited to mitigating relapse upon release from rehabilitation facilities and to overdose rescue than are the vaccines. Clearly, the active and passive immunotherapy approaches could be used together in individual patients. As there appears to be a minimum concentration of high-affinity anti-cocaine antibodies in order to be effective in relapse prevention, in those patients that produce sub-therapeutic concentrations the anti-cocaine mAb would augment their cocaine binding capacity to above therapeutic concentrations.