Utilizing surface plasmon resonance as a novel method for monitoring in-vitro P-glycoprotein efflux.

P-glycoprotein (Pgp) is known for its dichotomous roles as both a safeguarding efflux transporter against xenobiotics and as a catalyst for multidrug resistance. Given the susceptibility of numerous therapeutic compounds to Pgp-mediated resistance, compliance with Food and Drug Administration (FDA) guidelines mandates an in-depth in vitro transport assay during drug development. This study introduces an innovative transport assay that aligns with these regulatory imperatives but also addresses limitations in the currently established techniques. Using Pgp-reconstituted liposomes and employing surface plasmon resonance (SPR), this study developed a distinct method of measuring the relative transport rates of Pgp substrates in a controlled microenvironment. The Pgp substrates selected for this study-quinidine, methadone, and desipramine-resulted in transport ratios that corroborate with trends previously observed. To assess the kinetics of Pgp-mediated transport, the results were analyzed by fitting the data to both currently proposed Pgp substrate translocation models-the vacuum cleaner and flippase models. While the resulting kinetic analysis in this study lends support predominantly to the vacuum cleaner model, this study most notably developed a novel method of assessing Pgp-mediated transport rates and real-time kinetics using surface plasmon resonance.

Probing the Mechanisms Underlying the Transport of the Vinca Alkaloids by P-glycoprotein.

The efficacy of many cancer drugs is hindered by P-glycoprotein (Pgp), a cellular pump that removes drugs from cells. To improve chemotherapy, drugs capable of evading Pgp must be developed. Despite similarities in structure, vinca alkaloids (VAs) show disparate Pgp-mediated efflux ratios. ATPase activity and binding affinity studies show at least two binding sites for the VAs: high- and low-affinity sites that stimulate and inhibit the ATPase activity rate, respectively. The affinity for ATP from the ATPase kinetics curve for vinblastine (VBL) at the high-affinity site was 2- and 9-fold higher than vinorelbine (VRL) and vincristine (VCR), respectively. Conversely, VBL had the highest Km (ATP) for the low-affinity site. The dissociation constants (KDs) determined by protein fluorescence quenching were in the order VBL < VRL< VCR. The order of the KDs was reversed at higher substrate concentrations. Acrylamide quenching of protein fluorescence indicate that the VAs, either at 10 µM or 150 µM, predominantly maintain Pgp in an open-outward conformation. When 3.2 mM AMPPNP was present, 10 µM of either VBL, VRL, or VCR cause Pgp to shift to an open-outward conformation, while 150 µM of the VAs shifted the conformation of Pgp to an intermediate orientation, between opened inward and open-outward. However, the conformational shift induced by saturating AMPPNP and VCR condition was less than either VBL or VRL in the presence of AMPPNP. At 150 µM, atomic force microscopy (AFM) revealed that the VAs shift Pgp population to a predominantly open-inward conformation. Additionally, STDD NMR studies revealed comparable groups in VBL, VRL, and VCR are in contact with the protein during binding. Our results, when coupled with VAs-microtubule structure-activity relationship studies, could lay the foundation for developing next-generation VAs that are effective as anti-tumor agents. A model that illustrates the intricate process of Pgp-mediated transport of the VAs is presented.

Drug-Induced Conformational Dynamics of P-Glycoprotein Underlies the Transport of Camptothecin Analogs.

P-glycoprotein (Pgp) plays a pivotal role in drug bioavailability and multi-drug resistance development. Understanding the protein's activity and designing effective drugs require insight into the mechanisms underlying Pgp-mediated transport of xenobiotics. In this study, we investigated the drug-induced conformational changes in Pgp and adopted a conformationally-gated model to elucidate the Pgp-mediated transport of camptothecin analogs (CPTs). While Pgp displays a wide range of conformations, we simplified it into three model states: 'open-inward', 'open-outward', and 'intermediate'. Utilizing acrylamide quenching of Pgp fluorescence as a tool to examine the protein's tertiary structure, we observed that topotecan (TPT), SN-38, and irinotecan (IRT) induced distinct conformational shifts in the protein. TPT caused a substantial shift akin to AMPPNP, suggesting ATP-independent 'open-outward' conformation. IRT and SN-38 had relatively moderate effects on the conformation of Pgp. Experimental atomic force microscopy (AFM) imaging supports these findings. Further, the rate of ATPase hydrolysis was correlated with ligand-induced Pgp conformational changes. We hypothesize that the separation between the nucleotide-binding domains (NBDs) creates a conformational barrier for substrate transport. Substrates that reduce the conformational barrier, like TPT, are better transported. The affinity for ATP extracted from Pgp-mediated ATP hydrolysis kinetics curves for TPT was about 2-fold and 3-fold higher than SN-38 and IRT, respectively. On the contrary, the dissociation constants (KD) determined by fluorescence quenching for these drugs were not significantly different. Saturation transfer double difference (STDD) NMR of TPT and IRT with Pgp revealed that similar functional groups of the CPTs are accountable for Pgp-CPTs interactions. Efforts aimed at modifying these functional groups, guided by available structure-activity relationship data for CPTs and DNA-Topoisomerase-I complexes, could pave the way for the development of more potent next-generation CPTs.

Advantages and potential limitations of applying AFM kymograph analysis to pharmaceutically relevant membrane proteins in lipid bilayers.

Membrane proteins play critical roles in disease and in the disposition of many pharmaceuticals. A prime example is P-glycoprotein (Pgp) which moves a diverse range of drugs across membranes and out of the cell before a therapeutic payload can be delivered. Conventional structural biology methods have provided a valuable framework for comprehending the complex conformational changes underlying Pgp function, which also includes ATPase activity, but the lack of real-time information hinders understanding. Atomic force microscopy (AFM) is a single-molecule technique that is well-suited for studying active membrane proteins in bilayers and is poised to advance the field beyond static snapshots. After verifying Pgp activity in surface-support bilayers, we used kymograph analysis in conjunction with AFM imaging and simulations to study structural transitions at the 100 ms timescale. Though kymographs are frequently employed to boost temporal resolution, the limitations of the method have not been well characterized, especially for sparse non-crystalline distributions of pharmaceutically relevant membrane proteins like Pgp. Common experimental challenges are analyzed, including protein orientation, instrument noise, and drift. Surprisingly, a lateral drift of 75% of the protein dimension leads to only a 12% probability of erroneous state transition detection; average dwell time error achieves a maximum value of 6%. Rotational drift of proteins like Pgp, with azimuthally-dependent maximum heights, can lead to artifactual transitions. Torsional constraints can alleviate this potential pitfall. Confidence in detected transitions can be increased by adding conformation-altering ligands such as non-hydrolysable analogs. Overall, the data indicate that AFM kymographs are a viable method to access conformational dynamics for Pgp, but generalizations of the method should be made with caution.

Four Decades of Cytochrome P450 2B Research: From Protein Adducts to Protein Structures and Beyond.

This article features selected findings from the senior author and colleagues dating back to 1978 and covering approximately three-fourths of the 60 years since the discovery of cytochrome P450. Considering the vast number of P450 enzymes in this amazing superfamily and their importance for so many fields of science and medicine, including drug design and development, drug therapy, environmental health, and biotechnology, a comprehensive review of even a single topic is daunting. To make a meaningful contribution to the 50th anniversary of Drug Metabolism and Disposition, we trace the development of the research in a single P450 laboratory through the eyes of seven individuals with different backgrounds, perspectives, and subsequent career trajectories. All co-authors are united in their fascination for the structural basis of mammalian P450 substrate and inhibitor selectivity and using such information to improve drug design and therapy. An underlying theme is how technological advances enable scientific discoveries that were impossible and even inconceivable to prior generations. The work performed spans the continuum from: 1) purification of P450 enzymes from animal tissues to purification of expressed human P450 enzymes and their site-directed mutants from bacteria; 2) inhibition, metabolism, and spectral studies to isothermal titration calorimetry, deuterium exchange mass spectrometry, and NMR; 3) homology models based on bacterial P450 X-ray crystal structures to rabbit and human P450 structures in complex with a wide variety of ligands. Our hope is that humanizing the scientific endeavor will encourage new generations of scientists to make fundamental new discoveries in the P450 field. SIGNIFICANCE STATEMENT: The manuscript summarizes four decades of work from Dr. James Halpert's laboratory, whose investigations have shaped the cytochrome P450 field, and provides insightful perspectives of the co-authors. This work will also inspire future drug metabolism scientists to make critical new discoveries in the cytochrome P450 field.

The Structure and Mechanism of Drug Transporters.

Drug transporters are integral membrane proteins that play a critical role in drug disposition by affecting absorption, distribution, and excretion. They translocate drugs, as well as endogenous molecules and toxins, across membranes using ATP hydrolysis, or ion/concentration gradients. In general, drug transporters are expressed ubiquitously, but they function in drug disposition by being concentrated in tissues such as the intestine, the kidneys, the liver, and the brain. Based on their primary sequence and their mechanism, transporters can be divided into the ATP-binding cassette (ABC), solute-linked carrier (SLC), and the solute carrier organic anion (SLCO) superfamilies. Many X-ray crystallography and cryo-electron microscopy (cryo-EM) structures have been solved in the ABC and SLC transporter superfamilies or of their bacterial homologs. The structures have provided valuable insight into the structural basis of transport. This chapter will provide particular focus on the promiscuous drug transporters because of their effect on drug disposition and the challenges associated with them.

Deglycosylated Azithromycin Targets Transgelin to Enhance Intestinal Smooth Muscle Function.

Azithromycin (AZM) has been widely used as an antibacterial drug for many years. It has also been used to treat delayed gastric emptying. However, it exerts several side effects. We found that deglycosylated AZM (Deg-AZM or CP0119), an AZM metabolite, is a positively strong intestinal agonist that may result in the intestinal mobility experienced by patients after AZM administration. We confirmed that Deg-AZM can function strongly on intestinal peristalsis and identified transgelin as its potential molecular target. Furthermore, our pharmacological studies showed that the binding of Deg-AZM to transgelin enhanced the contractility of intestinal smooth muscle cells by facilitating the assembly of actin filaments into tight bundles and stress fibers. Specifically, Deg-AZM promoted intestinal peristaltic activity in wild-type mice but not in transgelin (-/-) mice. Moreover, Deg-AZM did not exert antibacterial activity and did not disrupt intestinal flora. Thus, Deg-AZM may become a potential drug for slow-transit constipation treatment.

Mechanisms and the clinical relevance of complex drug-drug interactions.

As a result of an increasing aging population, the number of individuals taking multiple medications simultaneously has grown considerably. For these individuals, taking multiple medications has increased the risk of undesirable drug-drug interactions (DDIs), which can cause serious and debilitating adverse drug reactions (ADRs). A comprehensive understanding of DDIs is needed to combat these deleterious outcomes. This review provides a synopsis of the pharmacokinetic (PK) and pharmacodynamic (PD) mechanisms that underlie DDIs. PK-mediated DDIs affect all aspects of drug disposition: absorption, distribution, metabolism and excretion (ADME). In this review, the cells that play a major role in ADME and have been investigated for DDIs are discussed. Key examples of drug metabolizing enzymes and drug transporters that are involved in DDIs and found in these cells are described. The effect of inhibiting or inducing these proteins through DDIs on the PK parameters is also reviewed. Despite most DDI studies being focused on the PK effects, DDIs through PD can also lead to significant and harmful effects. Therefore, this review outlines specific examples and describes the additive, synergistic and antagonistic mechanisms of PD-mediated DDIs. The effects DDIs on the maximum PD response (E max) and the drug dose or concentration (EDEC50) that lead to 50% of E max are also examined. Significant gaps in our understanding of DDIs remain, so innovative and emerging approaches are critical for overcoming them.

A Conformationally Gated Model of Methadone and Loperamide Transport by P-Glycoprotein.

P-glycoprotein (Pgp) is a multidrug resistance transporter that limits the penetration of a wide range of neurotherapeutics into the brain including opioids. The diphenylpropylamine opioids methadone and loperamide are structurally similar, but loperamide has about a 4-fold higher Pgp-mediated transport rate. In addition to these differences, they showed significant differences in their effects on Pgp-mediated adenosine triphosphate (ATP) hydrolysis. The activation of Pgp-mediated ATP hydrolysis by methadone was monophasic, whereas loperamide activation of ATP hydrolysis was biphasic implying methadone has a single binding site and loperamide has 2 binding sites on Pgp. Quenching of tryptophan fluorescence with these drugs and digoxin showed competition between the opioids and that loperamide does not compete for the digoxin-binding site. Acrylamide quenching of tryptophan fluorescence to probe Pgp conformational changes revealed that methadone- and loperamide-induced conformational changes were distinct. These results were used to develop a model for Pgp-mediated transport of methadone and loperamide where opioid binding and conformational changes are used to explain the differences in the opioid transport rates between methadone and loperamide.

Insights Into the Molecular Mechanism of Triptan Transport by P-glycoprotein.

The P-glycoprotein (Pgp) transporter reduces the penetration of a chemically diverse range of neurotherapeutics at the blood-brain barrier, but the molecular features of drugs and drug-Pgp interactions that drive transport remain to be clarified. In particular, the triptan neurotherapeutics, eletriptan (ETT) and sumatriptan (STT), were identified to have a >10-fold difference in transport rates despite being from the same drug class. Consistent with these transport differences, ETT activated Pgp-mediated ATP hydrolysis ∼2-fold, whereas STT slightly inhibited Pgp-mediated ATP hydrolysis by ∼10%. The interactions between them were also noncompetitive, suggesting that they occupy different binding sites on the transporter. Despite these differences, protein fluorescence spectroscopy revealed that the drugs have similar affinity to the transporter. NMR with Pgp and the drugs showed that they have distinct interactions with the transporter. Tertiary conformational changes probed by acrylamide quenching of Pgp tryptophan fluorescence with the drugs and a nonhydrolyzable ATP analog implied that the STT-bound Pgp must undergo larger conformational changes to hydrolyze ATP than ETT-bound Pgp. These results and previous transport studies were used to build a conformationally driven model for triptan transport with Pgp where STT presents a higher conformational barrier for ATP hydrolysis and transport than ETT.

Cardiovascular Ion Channel Inhibitor Drug-Drug Interactions with P-glycoprotein.

P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that plays a major role in cardiovascular drug disposition by effluxing a chemically and structurally diverse range of cardiovascular therapeutics. Unfortunately, drug-drug interactions (DDIs) with the transporter have become a major roadblock to effective cardiovascular drug administration because they can cause adverse drug reactions (ADRs) or reduce the efficacy of drugs. Cardiovascular ion channel inhibitors are particularly susceptible to DDIs and ADRs with Pgp because they often have low therapeutic indexes and are commonly coadministered with other drugs that are also Pgp substrates. DDIs from cardiovascular ion channel inhibitors with the transporter occur because of inhibition or induction of the transporter and the transporter's tissue and cellular localization. Inhibiting Pgp can increase absorption and reduce excretion of drugs, leading to elevated drug plasma concentrations and drug toxicity. In contrast, inducing Pgp can have the opposite effect by reducing the drug plasma concentration and its efficacy. A number of in vitro and in vivo studies have already demonstrated DDIs from several cardiovascular ion channel inhibitors with human Pgp and its animal analogs, including verapamil, digoxin, and amiodarone. In this review, Pgp-mediated DDIs and their effects on pharmacokinetics for different categories of cardiovascular ion channel inhibitors are discussed. This information is essential for improving pharmacokinetic predictions of cardiovascular therapeutics, for safer cardiovascular drug administration and for mitigating ADRs emanating from Pgp.

Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein.

The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis.

The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides.

BACKGROUND: Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure. METHODS: The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures. RESULTS: Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F-G loop regions and active site cavity volumes for the F87A mutated structures. CONCLUSIONS: Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site. GENERAL SIGNIFICANCE: The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.

Unravelling the complex drug-drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein.

Drug-drug interactions (DDIs) and associated toxicity from cardiovascular drugs represents a major problem for effective co-administration of cardiovascular therapeutics. A significant amount of drug toxicity from DDIs occurs because of drug interactions and multiple cardiovascular drug binding to the efflux transporter P-glycoprotein (Pgp), which is particularly problematic for cardiovascular drugs because of their relatively low therapeutic indexes. The calcium channel antagonist, verapamil and the cardiac glycoside, digoxin, exhibit DDIs with Pgp through non-competitive inhibition of digoxin transport, which leads to elevated digoxin plasma concentrations and digoxin toxicity. In the present study, verapamil-induced ATPase activation kinetics were biphasic implying at least two verapamil-binding sites on Pgp, whereas monophasic digoxin activation of Pgp-coupled ATPase kinetics suggested a single digoxin-binding site. Using intrinsic protein fluorescence and the saturation transfer double difference (STDD) NMR techniques to probe drug-Pgp interactions, verapamil was found to have little effect on digoxin-Pgp interactions at low concentrations of verapamil, which is consistent with simultaneous binding of the drugs and non-competitive inhibition. Higher concentrations of verapamil caused significant disruption of digoxin-Pgp interactions that suggested overlapping and competing drug-binding sites. These interactions correlated to drug-induced conformational changes deduced from acrylamide quenching of Pgp tryptophan fluorescence. Also, Pgp-coupled ATPase activity kinetics measured with a range of verapamil and digoxin concentrations fit well to a DDI model encompassing non-competitive and competitive inhibition of digoxin by verapamil. The results and previous transport studies were combined into a comprehensive model of verapamil-digoxin DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes.

Cross-linking mass spectrometry and mutagenesis confirm the functional importance of surface interactions between CYP3A4 and holo/apo cytochrome b(5).

Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.

Stereoselective formation and metabolism of 4-hydroxy-retinoic Acid enantiomers by cytochrome p450 enzymes.

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the ß-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.

The structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human cytochrome P450 3A4.

Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1'-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1'-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T(1) nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T(1) NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ molecules or an MDZ molecule and a CBZ molecule assume a stacked configuration within the CYP3A4 active site. In either case, the proton at position 4 of the MDZ molecule was closer to the heme than the protons of the 1'-CH(3) group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1'-CH(3) position closer to the heme than position 4. This study provides the first detailed molecular analysis of heterotropic and homotropic cooperativity of a human cytochrome P450 from an NMR-based model. Cooperativity of ligand binding through direct interaction between stacked molecules may represent a common motif for homotropic and heterotropic cooperativity.

Structural analysis of mammalian cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene: insight into partial enzymatic activity.

A combined structural and computational analysis of rabbit cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene (tBPA) has yielded insight into how the enzyme retains partial activity. Since conjugation to tBPA modifies a highly conserved active site residue, the residual activity of tBPA-labeled 2B4 observed in previous studies was puzzling. Here we describe the first crystal structures of a modified mammalian P450, which show an oxygenated metabolite of tBPA conjugated to Thr 302 of helix I. These results are consistent with previous studies that identified Thr 302 as the site of conjugation. In each structure, the core of 2B4 remains unchanged, but the arrangement of plastic regions differs. This results in one structure that is compact and closed. In this conformation, tBPA points toward helix B', making a 31° angle with the heme plane. This conformation is in agreement with previously performed in silico experiments. However, dimerization of 2B4 in the other structure, which is caused by movement of the B/C loop and helices F through G, alters the position of tBPA. In this case, tBPA lies almost parallel to the heme plane due to the presence of helix F' of the opposite monomer entering the active site to stabilize the dimer. However, docking experiments using this open form show that tBPA is able to rotate upward to give testosterone and 7-ethoxy-4-trifluoromethylcoumarin access to the heme, which could explain the previously observed partial activity.

NMR-derived models of amidopyrine and its metabolites in complexes with rabbit cytochrome P450 2B4 reveal a structural mechanism of sequential N-dealkylation.

To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T(1)) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R(M)) determined from T(1) relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, we used distances calculated from the relaxation rates to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyls are free to rotate between two binding modes. From these bound structures, a model of N-demethylation in which the N-linked methyl functional groups rotate between catalytic and noncatalytic positions was developed. This study is the first to provide a structural model of a drug and its metabolites complexed to a cytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine.

Yeast-hybrid based high-throughput assay for identification of anthrax lethal factor inhibitors.

Inhibitors of anthrax lethal factor (LF) are currently being sought as effective therapeutics for the treatment of anthrax. Here we report a novel screening approach for inhibitors of LF, a yeast-hybrid-based assay system in which the expression of reporter genes from a Gal4 promoter is repressed by LF proteolytic activity. Yeast cells were co-transformed with LF and a chimeric transcription factor that contains an LF substrate sequence inserted between the DNA-binding and activation domains of Gal4. In the resulting yeast cells, LF cleaves the substrate, thus inactivating the chimeric Gal4 and resulting in lack of expression of reporter genes. Compounds that inhibit LF cleavage of its substrate are identified by changes in reporter gene activity. Relative to in vitro screens for inhibitors of LF proteolytic activity, this screen has the advantage of excluding compounds that are toxic or non-permeable to eukaryotic cells. Additionally, the screen has the advantage of being fast, easy and cheap because exogenous LF and substrate are not needed. An initial chemical library screen with this system has identified four candidate inhibitors which were confirmed to inhibit LF protease activity in an in vitro assay. Furthermore, FBS-00831, one of the compounds identified, protects Raw 264.7 macrophages from anthrax lethal toxin and the possible binding site on LF was also evaluated by molecular docking.