Long Range Communication between the Drug-Binding Sites and Nucleotide Binding Domains of the Efflux Transporter ABCB1.

The ABC efflux pump P-glycoprotein (P-gp) transports a wide variety of drugs and is inhibited by others. Some drugs stimulate ATP hydrolysis at the nucleotide binding domains (NBDs) and are transported, others uncouple ATP hydrolysis and transport, and others inhibit ATP hydrolysis. The molecular basis for the different behavior of these drugs is not well understood despite the availability of several structural models of P-gp complexes with ligands bound. Hypothetically, ligands differentially alter the conformational dynamics of peptide segments that mediate the coupling between the drug binding sites and the NBDs. Here, we explore by hydrogen-deuterium exchange mass spectrometry the dynamic consequences of a classic substrate and inhibitor, vinblastine and zosuquidar, binding to mouse P-gp (mdr1a) in lipid nanodiscs. The dynamics of P-gp in nucleotide-free, pre-hydrolysis, and post-hydrolysis states in the presence of each drug reveal distinct mechanisms of ATPase stimulation and implications for transport. For both drugs, there are common regions affected in a similar manner, suggesting that particular networks are the key to stimulating ATP hydrolysis. However, drug binding effects diverge in the post-hydrolysis state, particularly in the intracellular helices (ICHs 3 and 4) and neighboring transmembrane helices. The local dynamics and conformational equilibria in this region are critical for the coupling of drug binding and ATP hydrolysis and are differentially modulated in the catalytic cycle.

Probing interactions of therapeutic antibodies with serum via second virial coefficient measurements.

Antibody-based therapeutics are the fastest-growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. Although the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small-molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, thereby altering therapeutic effect and disposition in ways that are incompletely understood. Although excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B2) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy is able to measure diffusive B2-values directly in undiluted serum. Apparent second virial coefficient (B2,app) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the approach of isolating specific components and completing independent cross-term virial coefficient measurements may not be an effective approach to characterizing nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general and advance the development of therapeutic antibodies and other protein-based therapeutics.

Cholesterol Asymmetrically Modulates the Conformational Ensemble of the Nucleotide-Binding Domains of P-Glycoprotein in Lipid Nanodiscs.

P-Glycoprotein (P-gp) is an ATP-dependent efflux pump that clears a wide variety of drugs and toxins from cells. P-gp undergoes large-scale structural changes and demonstrates conformational heterogeneity even within a single catalytic or drug-bound state, although the role of heterogeneity remains unclear. P-gp is found in a variety of cell types that vary in lipid composition, which modulates its activity. An understanding of structural or dynamic changes due to the lipid environment is lacking. We aimed to determine the effects of cholesterol in a membrane on the conformational behavior of P-gp in lipid nanodiscs. The presence of cholesterol stimulates ATP hydrolysis and alters lipid order and fluidity. Hydrogen/deuterium exchange mass spectrometry demonstrates that cholesterol in the membrane induces asymmetric, long-range changes in the distributions and exchange kinetics of conformations of the nucleotide-binding domains, correlating the effects of lipid composition on activity with specific changes in the P-gp conformational landscape.

Analytical and functional aspects of protein-ligand interactions: Beyond induced fit and conformational selection.

Ligand-dependent changes in protein conformation are foundational to biology. Historical mechanistic models for substrate-specific proteins are induced fit (IF) and conformational selection (CS), which invoke a change in protein conformation after ligand binds or before ligand binds, respectively. These mechanisms have important, but rarely discussed, functional relevance because IF vs. CS can differentially affect a protein's substrate specificity or promiscuity, and its regulatory properties. The modern view of proteins as conformational ensembles in both ligand free and bound states, together with the realization that most proteins exhibit some substrate promiscuity, demands a deeper interpretation of the historical models and provides an opportunity to improve mechanistic analyses. Here we describe alternative analytical strategies for distinguishing the historical models, including the more complex expanded versions of IF and CS. Functional implications of the different models are described. We provide an alternative perspective based on protein ensembles interacting with ligand ensembles that clarifies how a single protein can 'apparently' exploit different mechanisms for different ligands. Mechanistic information about protein ensembles can be optimized when they are probed with multiple ligands.

Dynamics and Location of the Allosteric Midazolam Site in Cytochrome P4503A4 in Lipid Nanodiscs.

Promiscuous and allosteric drug interactions with cytochrome P450 3A4 (CYP3A4) are ubiquitous but incompletely understood at the molecular level. A classic allosteric CYP3A4 drug interaction includes the benzodiazepine midazolam (MDZ). MDZ exhibits homotropic and heterotropic allostery when metabolized to 1'-hydroxy and 4-hydroxy metabolites in varying ratios. The combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Gaussian accelerated molecular dynamics (GaMD) simulations of CYP3A4 in lipid nanodiscs and in a lipid bilayer, respectively, reveals MDZ-dependent changes in dynamics in a membrane environment. The F-, G-, and intervening helices, as well as the loop preceding the ß1-sheets, display the largest observed changes in HDX. The GaMD suggests a potential allosteric binding site for MDZ in the F'- and G'-regions, which undergo significant increases in HDX at near-saturating MDZ concentrations. The HDX-MS and GaMD results confirm that changes in dynamics are most significant near the developing consensus allosteric site, and these changes are distinct from those observed previously with the nonallosteric inhibitor ketoconazole. The results suggest that the allosteric MDZ remains mobile in its binding site at the Phe-cluster. The results further suggest that this binding site remains dynamic or changes the depth of insertion in the membrane.

Dynamics and Mechanism of Binding of Androstenedione to Membrane-Associated Aromatase.

Aromatase (CYP19A1) catalyzes the synthesis of estrogens from androgens and is an invaluable target of pharmacotherapy for estrogen-dependent cancers. CYP19A1 is also one of the most primordial human CYPs and, to the extent that its fundamental dynamics are conserved, is highly relevant to understanding those of the more recently evolved and promiscuous enzymes. A complementary approach employing molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to interrogate the changes in CYP19A1 dynamics coupled to binding androstenedione (ASD). Gaussian-accelerated molecular dynamics and HDX-MS agree that ASD globally suppresses CYP19A1 dynamics. Bimodal HDX patterns of the B'-C loop potentially arising from at least two conformations are present in free 19A1 only, supporting the possibility that conformational selection is operative. Random-acceleration molecular dynamics and adaptive biasing force simulations illuminate ASD's binding pathway, predicting ASD capture in the lipid headgroups and a pathway to the active site shielded from solvent. Intriguingly, the predicted access channel in 19A1 aligns well with the steroid binding sites of other human sterol-oxidizing CYPs.

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors.

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel "2Fc" mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale.

P-glycoprotein (P-gp) is a highly substrate-promiscuous efflux transporter that plays a critical role in drug disposition. P-gp utilizes ATP hydrolysis by nucleotide-binding domains (NBDs) to drive transitions between inward-facing (IF) conformations that bind drugs and outward-facing (OF) conformations that release them to the extracellular solution. However, the details of the protein dynamics within either macroscopic IF or OF conformation remain uncharacterized, and the functional role of local dynamics has not been determined. In this work we measured the local dynamics of the IF state of P-gp in lipid nanodiscs and in detergent solution by hydrogen-deuterium (H/D) exchange MS. We observed "EX1 exchange kinetics," or bimodal kinetics, for several peptides distributed in both NBDs, particularly for P-gp in the lipid nanodiscs. Remarkably, the EX1 kinetics occurred on several time scales, ranging from seconds to hours, suggesting highly complex, and correlated, motions. The results indicate at least three distinct conformational states in the ligand-free P-gp and suggest a rough conformational landscape. Addition of excess ATP and vanadate, to favor the OF conformations, caused a generalized, but modest, decrease in H/D exchange throughout the NBDs and slowed the EX1 kinetic transitions of several peptides. The functional implications of the results are consistent with the possibility that conformational selection provides a source of substrate promiscuity.

Kinetic mechanism of controlled Fab-arm exchange for the formation of bispecific immunoglobulin G1 antibodies.

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Förster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the CH3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reaction.

Toward a Combinatorial Approach for the Prediction of IgG Half-Life and Clearance.

The serum half-life and clearance of therapeutic monoclonal antibodies (mAbs) are critical factors that impact their efficacy and optimal dosing regimen. The pH-dependent binding of an mAb to the neonatal Fc receptor (FcRn) has long been recognized as an important determinant of its pharmacokinetics. However, FcRn affinity alone is not a reliable predictor of mAb half-life, suggesting that other biologic or biophysical mechanisms must be accounted for. mAb thermal stability, which reflects its unfolding and aggregation propensities, may also relate to its pharmacokinetic properties. However, no rigorous statistical regression methods have been used to identify combinations of physical parameters that best predict biologic properties. In this work, a panel of eight mAbs with published human pharmacokinetic data were selected for biophysical analyses of FcRn binding and thermal stability. Biolayer interferometry was used to characterize FcRn/mAb binding at acidic and neutral pH, while differential scanning calorimetry was used to determine thermodynamic unfolding parameters. Individual binding or stability parameters were generally weakly correlated with half-life and clearance values. Least absolute shrinkage and selection operator regression was used to identify the combination of two parameters with the best correlation to half-life and clearance as being the FcRn binding response at pH 7.0 and the change in heat capacity. Leave-one-out subsampling yielded a root mean square difference between observed and predicted half-life of just 2.7 days (16%). Thus, the incorporation of multiple biophysical parameters into a cohesive model may facilitate early-stage prediction of in vivo half-life and clearance based on simple in vitro experiments.

Differential Coupling of Binding, ATP Hydrolysis, and Transport of Fluorescent Probes with P-Glycoprotein in Lipid Nanodiscs.

The ATP binding cassette transporter P-glycoprotein (ABCB1 or P-gp) plays a major role in cellular resistance to drugs and drug interactions. Experimental studies support a mechanism with nucleotide-dependent fluctuation between inward-facing and outward-facing conformations, which are coupled to nucleotide hydrolysis. However, detailed insight into drug-dependent modulation of these conformational ensembles is lacking. Different drugs likely occupy partially overlapping but distinct sites and are therefore variably coupled to nucleotide binding and hydrolysis. Many fluorescent drug analogues are used in cell-based transport models; however, their specific interactions with P-gp have not been studied, and this limits interpretation of transport assays in terms of molecular models. Here we monitor binding of the fluorescent probe substrates BODIPY-verapamil, BODIPY-vinblastine, and Flutax-2 at low occupancy to murine P-gp in lipid nanodiscs via fluorescence correlation spectroscopy, in variable nucleotide-bound states. Changes in affinity for the different nucleotide-dependent conformations are probe-dependent. For BODIPY-verapamil and BODIPY-vinblastine, there are 2-10-fold increases in KD in the nucleotide-bound or vanadate-trapped state, compared to that in the nucleotide-free state. In contrast, the affinity of Flutax-2 is unaffected by nucleotide or vanadate trapping. In further contrast to BODIPY-verapamil and BODIPY-vinblastine, Flutax-2 does not cause stimulation of ATP hydrolysis despite the fact that it is transported in vesicle-based transport assays. Whereas the established substrates verapamil, paclitaxel, and vinblastine displace BODIPY-verapamil or BODIPY-vinblastine from their high-affinity sites, the transport substrate Flutax-2 is not displaced by any of these substrates. The results demonstrate a unique binding site for Flutax-2 that allows for transport without stimulation of ATP hydrolysis.

Diffusion of Soluble Aggregates of THIOMABs and Bispecific Antibodies in Serum.

Submicrometer aggregates are frequently present at low levels in antibody-based therapeutics. Although intuition suggests that the fraction of the aggregate or the size of the aggregate present might correlate with deleterious clinical properties or formulation difficulties, it has been challenging to demonstrate which aggregate states, if any, trigger specific biological effects. One source of uncertainty about the putative linkage between aggregation and safety or efficacy lies in the likelihood that noncovalent aggregation differs in ideal buffers versus in serum and biological tissues; self-association or association with other proteins may vary widely with environment. Therefore, methods for monitoring aggregation and aggregate behavior in biologically relevant matrices could provide a tool for better predicting aggregate-dependent clinical outcomes and provide a basis for antibody engineering prior to clinical studies. Here, we generate models for soluble aggregates of THIOMABs and a bispecific antibody (bsAb) of defined size and exploit fluorescence correlation spectroscopy to monitor their diffusion properties in serum and viscosity-matched buffers. The monomers, dimers, and trimers of both THIOMABs and a bsAb reveal a modest increase in diffusion time in serum greater than expected for an increase in viscosity alone. A mixture of larger aggregates containing mostly bsAb pentamers exhibits a marked increase in diffusion time in serum and much greater intrasample variability, consistent with significant aggregation or interactions with serum components. The results indicate that small aggregates of several IgG platforms are not likely to aggregate with serum components, but nanometer-scale aggregates larger than trimers can interact with the serum in an Ab-dependent manner.

The Myeloablative Drug Busulfan Converts Cysteine to Dehydroalanine and Lanthionine in Redoxins.

The myeloablative agent busulfan (1,4-butanediol dimethanesulfonate) is an old drug that is used routinely to eliminate cancerous bone marrow prior to hematopoietic stem cell transplant. The myeloablative activity and systemic toxicity of busulfan have been ascribed to its ability to cross-link DNA. In contrast, here we demonstrate that incubation of busulfan with the thiol redox proteins glutaredoxin or thioredoxin at pH 7.4 and 37 °C results in the formation of putative S-tetrahydrothiophenium adducts at their catalytic Cys residues, followed by ß-elimination to yield dehydroalanine. Both proteins contain a second Cys, in their catalytic C-X-X-C motif, which reacts with the dehydroalanine, the initial Cys adduct with busulfan, or the S-tetrahydrothiophenium, to form novel intramolecular cross-links. The reactivity of the dehydroalanine (DHA) formed is further demonstrated by adduction with glutathione to yield a lanthionine and by a novel reaction with the reducing agent tris(2-carboxyethyl)phosphine (TCEP), which yields a phosphine adduct via Michael addition to the DHA. Formation of a second quaternary organophosphonium salt via nucleophilic substitution with TCEP on the initial busulfan-protein adduct or on the THT(+)-Redoxin species is also observed. These results reveal a rich potential for reactions of busulfan with proteins in vitro, and likely in vivo. It is striking that several of the chemically altered protein products retain none of the atoms of busulfan, in contrast to typical drug-protein adducts or traditional protein modification reagents. In particular, the ability of a clinically used drug to convert Cys to dehydrolanine in intact proteins, and its subsequent reaction with biological thiols, is unprecedented.

Membrane Fluidity Modulates Thermal Stability and Ligand Binding of Cytochrome P4503A4 in Lipid Nanodiscs.

Cytochrome P4503A4 (CYP3A4) is a peripheral membrane protein that plays a major role in enzymatic detoxification of many drugs and toxins. CYP3A4 has an integral membrane N-terminal helix and a localized patch comprised of the G' and F' helix regions that are embedded in the membrane, but the effects of membrane composition on CYP3A4 function are unknown. Here, circular dichroism and differential scanning calorimetry were used to compare the stability of CYP3A4 in lipid bilayer nanodiscs with varying ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These lipids differ in the acyl-chain length and their degree of unsaturation. The thermal denaturation of CYP3A4 in nanodiscs occurs in a temperature range distinct from that of the nanodisc denaturation so it can be monitored calorimetrically. Melting temperatures (Tm), heat capacities (ΔCp), and calorimetric enthalpies (ΔHcal) for denaturation of CYP3A4 each increased with an increasing fraction of DMPC, with a maximum at 50% DMPC, before decreasing at 75% DMPC. Addition of the inhibitor ketoconazole results in increased thermal stability, and larger ΔCp and ΔHcal values, with different sensitivities to lipid composition. Effects of lipid composition on ligand binding dynamics were also studied. Equilibrium binding affinities of both ketoconazole (KTZ) and testosterone (TST) were minimally affected by lipid composition. However, stopped-flow analyses indicate that the rates of KTZ binding reach a maximum in membranes containing 50% DMPC, whereas the rate of TST binding decreases continuously with an increasing DMPC concentration. These results indicate that CYP3A4 is highly sensitive to the acyl-chain composition of the lipids and fluidity of the membrane in which it is embedded.

Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs.

Membrane-bound cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug metabolism. Although several structural models of CYP3A4 in various ligand complexes are available, none includes a lipid bilayer. Details of the effects of the membrane on protein dynamics and solvation, and access channels for ligands, remain uncertain. H/D exchange mass spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion of residues 3-12, compared to that of the full length wild type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium exchange was fast in the F- and G-helices, HI loop, and C-terminal loop. In contrast, there is very low exchange in the F'- and G'-helices. The results are consistent with the overall membrane orientation of CYP3A4 suggested by published MD simulations and spectroscopic results, and the solvent accessibility of the F/G loop suggests that it is not deeply membrane-embedded. Addition of ketoconazole results in only modest, but global, changes in solvent accessibility. Interestingly, with ketoconazole bound some peptides become less solvent accessible or dynamic, including the F- and G-helices, but several peptides demonstrate modestly increased accessibility. Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests membrane-induced stabilization compared to that of aggregated CYP3A4 in buffer, and this stabilization is enhanced upon addition of the ligand ketoconazole. This ligand-induced stabilization is accompanied by a very large increase in ΔH for CYP3A4 denaturation in nanodiscs, possibly due to increased CYP3A4-membrane interactions. Together, the results suggest a distinct orientation of CYP3A4 on the lipid membrane, and they highlight likely solvent access channels, which are consistent with several MD simulations.

Drug modulation of water-heme interactions in low-spin P450 complexes of CYP2C9d and CYP125A1.

Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP-inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenylpropyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of "classic" type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A (15)N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution.

Comparison of epsilon- and delta-class glutathione S-transferases: the crystal structures of the glutathione S-transferases DmGSTE6 and DmGSTE7 from Drosophila melanogaster.

Cytosolic glutathione transferases (GSTs) comprise a large family of enzymes with canonical structures that diverge functionally and structurally among mammals, invertebrates and plants. Whereas mammalian GSTs have been characterized extensively with regard to their structure and function, invertebrate GSTs remain relatively unstudied. The invertebrate GSTs do, however, represent potentially important drug targets for infectious diseases and agricultural applications. In addition, it is essential to fully understand the structure and function of invertebrate GSTs, which play important roles in basic biological processes. Invertebrates harbor delta- and epsilon-class GSTs, which are not found in other organisms. Drosophila melanogaster GSTs (DmGSTs) are likely to contribute to detoxication or antioxidative stress during development, but they have not been fully characterized. Here, the structures of two epsilon-class GSTs from Drosophila, DmGSTE6 and DmGSTE7, are reported at 2.1 and 1.5 Šresolution, respectively, and are compared with other GSTs to identify structural features that might correlate with their biological functions. The structures of DmGSTE6 and DmGSTE7 are remarkably similar; the structures do not reveal obvious sources of the minor functional differences that have been observed. The main structural difference between the epsilon- and delta-class GSTs is the longer helix (A8) at the C-termini of the epsilon-class enzymes.

The busulfan metabolite EdAG irreversibly glutathionylates glutaredoxins.

The DNA alkylating agent busulfan is used to 'precondition' patients with leukemia, lymphomas and other hematological disorders prior to hematopoietic stem cell transplants. Busulfan is metabolized via conjugation with glutathione (GSH) followed by intramolecular rearrangement to the GSH analog γ-glutamyl-dehydroalanyl -glycine (EdAG). EdAG contains the electrophilic dehydroalanine, which is expected to react with protein nucleophiles, particularly proteins with GSH binding sites such as glutaredoxins (Grx's). Incubation of EdAG with human Grx-1 or Grx-2 results in facile adduction of cys-23 and cys-77, respectively, as determined by ESI-MS/MS. The resulting modified proteins are catalytically inactive. In contrast, the glutathione transferase A1-1 includes a GSH binding site with a potentially reactive tyrosinate (Tyr-9) but it does not react with EdAG. Similarly, Cys-112 of GSTA1-1, which lies outside the active site and is known to form disulfides with GSH, does not react with EdAG. The results provide the first demonstration of the reactivity of any busulfan metabolites with intact proteins, and they suggest that GSH-binding sites containing thiolates are most susceptible. The adduction of Grx's by EdAG suggests the possible alteration of proteins that are normally regulated via Grx-dependent reversible glutathionylation or deglutathionylation. Dysregulation of Grx-dependent processes could contribute to cellular toxicity of busulfan.

Reaction dynamics of ATP hydrolysis catalyzed by P-glycoprotein.

P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug-drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ(18)O4-labeled ATP, no positional isotope exchange is detectable at the bridging ß-phosphorus-O-γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three (18)O/two (18)O/one (18)O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO4(2-) (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states.