Utilizing surface plasmon resonance as a novel method for monitoring 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 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.

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.

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).

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.

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 , 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.

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.

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.

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.

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.

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%.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Structural features of cytochromes P450 and ligands that affect drug metabolism as revealed by X-ray crystallography and NMR.

Cytochromes P450 (P450s) play a major role in the clearance of drugs, toxins, and environmental pollutants. Additionally, metabolism by P450s can result in toxic or carcinogenic products. The metabolism of pharmaceuticals by P450s is a major concern during the design of new drug candidates.

Plasticity of cytochrome P450 2B4 as investigated by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography.

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI).

Intramolecular heme ligation of the cytochrome P450 2C9 R108H mutant demonstrates pronounced conformational flexibility of the B-C loop region: implications for substrate binding.

A previous study [Dickmann, L., et al. (2004) Mol.

Structures of cytochrome P450 2B4 complexed with the antiplatelet drugs ticlopidine and clopidogrel .

Prior X-ray crystal structures of rabbit cytochrome P450 2B4 (2B4) in complexes with various imidazoles have demonstrated markedly different enzyme conformations depending on the size of the inhibitor occupying the active site. In this study, structures of 2B4 were determined with the antiplatelet drugs clopidogrel and ticlopidine, which were expected to have greater freedom of movement in the binding pocket. Ticlopidine could be modeled into the electron density maps in two distinct orientations, both of which are consistent with metabolic data gathered with other mammalian P450 enzymes.

Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization.