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.

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.

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.

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.

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.

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. Determining the interactions between P450s and compounds of very diverse structures is complicated by the variability in P450-ligand interactions. Understanding the protein structural elements and the chemical attributes of ligands that dictate their orientation in the P450 active site will aid in the development of effective and safe therapeutic agents. The goal of this review is to describe P450-ligand interactions from two perspectives. The first is the various structural elements that microsomal P450s have at their disposal to assume the different conformations observed in X-ray crystal structures. The second is P450-ligand dynamics analyzed by NMR relaxation studies.

The stereochemical course of 4-hydroxy-2-nonenal metabolism by glutathione S-transferases.

4-Hydroxy-2-nonenal (HNE) is a toxic aldehyde generated during lipid peroxidation and has been implicated in a variety of pathological states associated with oxidative stress. Glutathione S-transferase (GST) A4-4 is recognized as one of the predominant enzymes responsible for the metabolism of HNE. However, substrate and product stereoselectivity remain to be fully explored. The results from a product formation assay indicate that hGSTA4-4 exhibits a modest preference for the biotransformation of S-HNE in the presence of both enantiomers. Liquid chromatography mass spectrometry analyses using the racemic and enantioisomeric HNE substrates explicitly demonstrate that hGSTA4-4 conjugates glutathione to both HNE enantiomers in a completely stereoselective manner that is not maintained in the spontaneous reaction. Compared with other hGST isoforms, hGSTA4-4 shows the highest degree of stereoselectivity. NMR experiments in combination with simulated annealing structure determinations enabled the determination of stereochemical configurations for the GSHNE diastereomers and are consistent with an hGSTA4-4-catalyzed nucleophilic attack that produces only the S-configuration at the site of conjugation, regardless of substrate chirality. In total these results indicate that hGSTA4-4 exhibits an intriguing combination of low substrate stereoselectivity with strict product stereoselectivity. This behavior allows for the detoxification of both HNE enantiomers while generating only a select set of GSHNE diastereomers with potential stereochemical implications concerning their effects and fates in biological tissues.

Cooperative binding of acetaminophen and caffeine within the P450 3A4 active site.

Acetaminophen (N-acetyl-p-aminophenol, APAP) is a commonly used analgesic/antipyretic. When oxidized by P450, a toxic APAP metabolite is generated. Human P450 3A4 was expressed in Escherichia coli , purified, and reconstituted using artificial liposomes. Oxidation of APAP by P450 3A4, as detected by the formation of its glutathione adduct, was found to exhibit negative homotropic cooperativity with a Hill coefficient of 0.7. In the presence of caffeine, the observed kinetics were close to classical Michaelis-Menten kinetics with a Hill coefficient approaching 1. In order to probe for a potential repositioning of APAP within the P450 3A4 pocket in the presence of caffeine, NMR T1 paramagnetic relaxation techniques were used to calculate distances from the P450 3A4 heme iron to protons of APAP alone and in the presence of caffeine. Both APAP and caffeine were found to bind at the active site in proximity to the heme iron. When APAP was incubated with P450 3A4, the acetamido group of APAP was found to be closest to the heme iron consistent with the amide group of APAP weakly associating with the heme iron. The addition of caffeine disrupted the ability of APAP to coordinate with the heme iron of P450 3A4 and enhanced the rate of oxidation to its toxic metabolite.

Cysteine 98 in CYP3A4 contributes to conformational integrity required for P450 interaction with CYP reductase.

Previously human cytochrome P450 3A4 was efficiently and specifically photolabeled by the photoaffinity ligand lapachenole. One of the modification sites was identified as cysteine 98 in the B-C loop region of the protein [B. Wen, C.E. Doneanu, C.A. Gartner, A.G. Roberts, W.M. Atkins, S.D. Nelson, Biochemistry 44 (2005) 1833-1845]. Loss of CO binding capacity and subsequent decrease of catalytic activity were observed in the labeled CYP3A4, which suggested that aromatic substitution on residue 98 triggered a critical conformational change and subsequent loss of enzyme activity. To test this hypothesis, C98A, C98S, C98F, and C98W mutants were generated by site-directed mutagenesis and expressed functionally as oligohistidine-tagged proteins. Unlike the mono-adduction observed in the wild-type protein, simultaneous multiple adductions occurred when C98F and C98W were photolabeled under the same conditions as the wild-type enzyme, indicating a substantial conformational change in these two mutants compared with the wild-type protein. Kinetic analysis revealed that the C98W mutant had a drastic 16-fold decrease in catalytic efficiency (V(max)/K(m)) for 1'-OH midazolam formation, and about an 8-fold decrease in catalytic efficiency (V(max)/K(m)) for 4-OH midazolam formation, while the C98A and C98S mutants retained the same enzyme activity as the wild-type enzyme. Photolabeling of C98A and C98S with lapachenole resulted in monoadduction of only Cys-468, in contrast to the labeling of Cys-98 in wild-type CYP3A4, demonstrating the marked selectivity of this photoaffinity ligand for cysteine residues. The slight increases in the midazolam binding constants (K(s)) in these mutants suggested negligible perturbation of the heme environment. Further activity studies using different P450:reductase ratios suggested that the affinity of P450 to reductase was significantly decreased in the C98W mutant, but not in the C98A and C98S mutants. In addition, the C98W mutant exhibited a 41% decrease in the maximum electron flow rate between P450 and reductase as measured by reduced nicotinamide adenine dinucleotide phosphate consumption at a saturating reductase concentration. In conclusion, our data strongly suggest that cysteine 98 in the B-C loop region significantly contributes to conformational integrity and catalytic activity of CYP3A4, and that this residue or residues nearby might be involved in an interaction with P450 reductase.

Probing the CYP3A4 active site by cysteine scanning mutagenesis and photoaffinity labeling.

The mechanism of CYP3A4-substrate interactions has been investigated using a battery of techniques including cysteine scanning mutagenesis, photoaffinity labeling, and structural modeling. In this study, cysteine scanning mutagenesis was performed at seven sites within CYP3A4 proposed to be involved in substrate interaction and/or cooperativity. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis for each mutant after proteolytic digestion and isolation of fluorescent photolabeled peptides. Among the tryptic peptides of seven tested mutants, three photolabeled peptides of the F108C mutant, ECYSVFTNR (positions 97-105), VLQNFSFKPCK (positions 459-469), and RPCGPVGFMK (positions 106-115) were identified by MALDI-TOF-MS and nano-LC/ESI QTOF MS. The site of modification was further localized to the substituted Cys-108 residue in the mutant peptide adduct RPCGPVGFMK (positions 106-115) by nano-LC/ESI QTOF MS/MS. In summary, we described a potentially useful method to study P450 active sites using a combination of cysteine scanning mutagenesis and photoaffinity labeling.

Acceptor and donor-side interactions of phenolic inhibitors in Photosystem II.

Certain phenolic compounds represent a distinct class of Photosystem (PS) II Q(B) site inhibitors. In this paper, we report a detailed study of the effects of 2,4,6-trinitrophenol (TNP) and other phenolic inhibitors, bromoxynil and dinoseb, on PS II energetics. In intact PS II, phenolic inhibitors bound to only 90-95% of Q(B) sites even at saturating concentrations. The remaining PS II reaction centers (5-10%) showed modified Q(A) to Q(B) electron transfer but were sensitive to urea/triazine inhibitors. The binding of phenolic inhibitors was 30- to 300-fold slower than the urea/triazine class of Q(B) site inhibitors, DCMU and atrazine. In the sensitive centers, the S(2)Q(A)(-) state was 10-fold less stable in the presence of phenolic inhibitors than the urea/triazine herbicides. In addition, the binding affinity of phenolic herbicides was decreased 10-fold in the S(2)Q(A)(-) state than the S(1)Q(A) state. However, removal of the oxygen-evolving complex (OEC) and associated extrinsic polypeptides by hydroxylamine (HA) washing abolished the slow binding kinetics as well as the destabilizing effects on the charge-separated state. The S(2)-multiline electron paramagnetic resonance (EPR) signal and the 'split' EPR signal, originating from the S(2)Y(Z) state showed no significant changes upon binding of phenolic inhibitors at the Q(B) site. We thus propose a working model where Q(A) redox potential is lowered by short-range conformational changes induced by phenolic inhibitor binding at the Q(B) niche. Long-range effects of HA-washing eliminate this interaction, possibly by allowing more flexibility in the Q(B) site.

A bacterial flavin reductase system reduces chromate to a soluble chromium(III)-NAD(+) complex.

Biological reduction of carcinogenic chromate has been extensively studied in eukaryotic cells partly because the reduction produces stable chromium(III)-DNA adducts, which are mutagenic. Microbial reduction of chromate has been studied for bioremediation purposes, but little is known about the reduction mechanism. In eukaryotic cells chromate is mainly reduced non-enzymatically by ascorbate, which is usually absent in bacterial cells. We have characterized the reduction of chromate by a flavin reductase (Fre) from Escherichia coli with flavins. The Fre-flavin system rapidly reduced chromate, whereas chemical reduction by NADH and glutathione was very slow. Thus, enzymatic chromate reduction is likely the dominant mechanism in bacterial cells. Furthermore, the end-product was a soluble and stable Cr(III)-NAD(+) complex, instead of Cr(III) precipitate. Since intracellularly generated Cr(III) forms adducts with DNA, protein, glutathione, and ascorbate in eukaryotic cells, we suggest that the produced Cr(III) is primarily complexed to NAD(+), DNA, and other cellular components inside bacteria.

Certain metal ions are inhibitors of cytochrome b6f complex 'Rieske' iron-sulfur protein domain movements.

Many current models of the Q cycle for the cytochrome (cyt) b6f and the cyt bc1 complexes incorporate 'Rieske' iron-sulfur protein (ISP) domain movements to gate electron transfer and to ensure high yields of proton shuttling. It was previously proposed that copper ions, which bind at a site distant from the quinol oxidase (Q(o)) site, inhibit plastoquinol (PQH2) binding by restraining the hydrophilic head domain of the ISP [Rao B. K., S., Tyryshkin, A. M., Roberts, A. G., Bowman, M. K., and Kramer, D. M. (1999) Biochemistry 38, 3285-3296]. The present work presents evidence that this is indeed the case for both copper ions and Zn2+, which appear to inhibit by similar mechanisms. Electron paramagnetic resonance (EPR) spectra show that Cu2+ and Zn2+ binding to the cyt b6f complex displaces the Q(o) site inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). At high concentrations, both DBMIB and Cu2+ or Zn2+ can bind simultaneously, altering the Rieske 2Fe2S cluster and Cu2+ EPR spectra, suggesting perturbations in their respective binding sites. Both Zn2+ and Cu1+ altered the orientations of the Rieske 2Fe2S cluster with respect to the membrane plane, but had no effect on that of the cyt b6 hemes. Cu2+ was found to change the orientation of the cyt f heme plane, consistent with binding on the cyt f protein. Within conservative constraints, the data suggest that the ISP is shifted into a position intermediate between the ISP(C) position, when the Q(o) site is unoccupied, and the ISP(B) position, when the Q(o) site is occupied by inhibitors such as DBMIB or stigmatellin. These results support the role of ISP domain movements in Q(o) site catalysis.