Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding

A 1.9-A molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B' and C, the N terminus of helix I, and the beta(4) region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F' and G' helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding.     

Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis

P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.     

Conformational analysis of human ATP-binding cassette transporter ABCB1 in lipid nanodiscs and inhibition by the antibodies MRK16 and UIC2

The human ATP-binding cassette (ABC) transporter, P-glycoprotein (P-gp; ABCB1), mediates the ATP-dependent efflux of a variety of drugs. As a result, P-gp plays a critical role in tumor cell drug resistance and the pharmacokinetic properties of most drugs. P-gp exhibits extraordinary substrate and inhibitor promiscuity, resulting in a wide range of possible drug-drug interactions. Inhibitory antibodies have long been considered as a possible strategy to modulate P-gp-dependent cancer cell drug resistance, and it is widely suggested that the antibodies MRK16 and UIC2 inhibit P-gp by capturing a single isoform and preventing flux through the catalytic cycle. Although the crystal structures of many bacterial whole transporters, as well as isolated nucleotide-binding domains, have been solved, high resolution structural data for mammalian ABC transporters are currently lacking. It has been extremely difficult to determine the detailed mechanism of transport of P-gp, in part because it is difficult to obtain purified protein in well defined lipid systems. Here we exploit surface plasmon resonance (SPR) to probe conformational changes associated with these intermediate states for P-gp in lipid bilayer nanodiscs. The results indicate that P-gp in nanodiscs undergoes functionally relevant ligand-dependent conformational changes and that previously described inhibitory antibodies bind to multiple nucleotide-bound states but not the ADP-VO(4)-trapped state, which mimics the post-hydrolysis state. The results also suggest that the substrate drug vinblastine is released at stages that precede or follow the post-hydrolysis ADP-PO(4)·P-gp complex.     

Unbiased simulations reveal the inward-facing conformation of the human serotonin transporter and Na(+) ion release

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 μs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na(+)-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport.     

Targeting drug-efflux pumps -- a pharmacoinformatic approach

In line with our studies on propafenone-type inhibitors of P-glycoprotein (P-gp), we applied several methods to approach virtual screening tools for identification of new P-gp inhibitors on one hand and the molecular basis of ligand-protein interaction on the other hand. For virtual screening, a combination of autocorrelation vectors and selforganising artificial neural networks proved extremely valuable in identifying P-gp inhibitors with structurally new scaffolds. For a closer view on the binding region for propafenone-type ligands we applied a combination of pharmacophore-driven photoaffinity labeling and protein homology modeling. On LmrA, a bacterial homologue of P-gp, we were able to identify distinct regions on transmembrane helices 3, 5 and 6 which show significant changes in the labeling pattern during different steps of the catalytic cycle.     

The flexibility of P-glycoprotein for its poly-specific drug binding from molecular dynamics simulations

The multidrug efflux pump P-glycoprotein (P-gp) contributes to multidrug resistance in about half of human cancers. Recently, high resolution X-ray crystal structures of mouse P-gp (inward-facing) were reported, which significantly facilitates the understanding of the function of P-gp and the structure-based design of inhibitors for P-gp. Here we perform 20?ns molecular dynamics simulations of inward-facing P-gp with/without ligand in explicit lipid and water to investigate the flexibility of P-gp for its poly-specific drug binding. By analyzing the interactions between P-gp and QZ59-RRR or QZ59-SSS, we summarize the important residues and the flexibility of different parts of P-gp. Particularly, the flexibility of the side chains of aromatic residues (Phe and Tyr) allows them to form rotamers with different orientations in the binding pocket, which plays a critical role for the poly-specificity of the drug-binding cavity of P-gp. MD simulations reveal that trans-membrane (TM) TM12 and TM6 are flexible and contribute to the poly-specific drug binding, while TM4 and TM5 are rigid and stabilize the whole structure. We also construct outward-facing P-gp based on the MsbA structure and perform 20?ns MD simulations. The comparison between the MD results for outward-facing P-gp and those for inward-facing P-gp shows that the TM parts in outward-facing P-gp undergo significant conformational change to facilitate the export of small molecules.     

Conformational sampling and nucleotide-dependent transitions of the GroEL subunit probed by unbiased molecular dynamics simulations

GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant "opening" of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition.     

P-Glycoprotein (P-gp) expressed in a confluent monolayer of hMDR1-MDCKII cells has more than one efflux pathway with cooperative binding sites

The multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of substrates and study inhibition using pairwise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single binding site, because a previous study proved that the mass-action kinetics of efflux of a single substrate were not sensitive to whether there are one or more substrate-binding and efflux sites. In this study, using this one-site model, we found that, with "high" concentrations of either a substrate or an inhibitor, the elementary rate constants fitted independently for each of the substrates alone quantitatively predicted the efflux curves, simply applying the assumption that binding at the "one site" was competitive. On the other hand, at "low" concentrations of both the substrate and inhibitor, we found no inhibition of the substrate efflux, despite the fact that both the substrate and inhibitor were being well-effluxed. This was not an effect of excess "empty" P-gp molecules, because the competitive efflux model takes site occupancy into account. Rather, it is quantitative evidence that the substrate and inhibitor are being effluxed by multiple pathways within P-gp. Remarkably, increasing the substrate concentration above the "low" concentration, caused the inhibition to become competitive; i.e., the inhibitor became effective. These data and their analysis show that the binding of these substrates must be cooperative, either positive or negative.     

Antisense oligonucleotides targeting universally conserved 26S rRNA domains of plant ribosomes at different steps of polypeptide elongation

A ribosome undergoes significant conformational changes during elongation of polypeptide chain that are correlated with structural changes of rRNAs. We tested nine different antisense oligodeoxynucleotides complementary to the selected, highly conserved sequences of Lupinus luteus 26S rRNA that are engaged in the interactions with tRNA molecules. The ribosomes were converted either to pre- or to posttranslocational states, with or without prehybridized oligonucleotides, using tRNA or mini-tRNA molecules. The activity of those ribosomes was tested via the so-called binding assay. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis. In this article, we present that (i) before and after translocation, fragments of domain V between helices H70/H71 and H74/H89 do not have to interact with nucleotides 72-76 of the acceptor arm of A-site tRNA; (ii) helix H69 does not have to interact with DHU arm of tRNA in positions 25 and 26 after forming the peptide bond, but before translocation; (iii) helices H69 and H70 interact weakly with nucleotides 11, 12, 25, and 26 of A-site tRNA before forming a peptide bond in the ribosome; (iv) interactions between helices H80, H93 and single-stranded region between helices H92 and H93 and CCAend of P-site tRNA are necessary at all steps of elongation cycle; and (v) before and after translocation, helix H89 does not have to interact with nucleotides in positions 64-65 and 50-53 of A-site tRNA TPsiC arm.     

Exploring movement and energy in human P-glycoprotein conformational rearrangement

Human P-glycoprotein (P-gp), a kind of ATP-Binding Cassette transporter, can export a diverse variety of anti-cancer drugs out of the tumor cell. Its overexpression is one of the main reasons for the multidrug resistance (MDR) of tumor cells. It has been confirmed that during the substrate transport process, P-gp experiences a large-scale structural rearrangement from the inward- to outward-facing states. However, the mechanism of how the nucleotide-binding domains (NBDs) control the transmembrane domains (TMDs) to open towards the periplasm in the outward-facing state has not yet been fully characterized. Herein, targeted molecular dynamics simulations were performed to explore the conformational rearrangement of human P-gp. The results show that the allosteric process proceeds in a coupled way, and first the transition is driven by the NBDs, and then transmitted to the cytoplasmic parts of TMDs, finally to the periplasmic parts. The trajectories show that besides the translational motions, the NBDs undergo a rotation movement, which mainly occurs in xy plane and ensures the formation of the correct ATP-binding pockets. The analyses on the interaction energies between the six structure segments (cICLs) from the TMDs and NBDs reveal that their subtle energy differences play an important role in causing the periplasmic parts of the transmembrane helices to separate from each other in the established directions and in appropriate amplitudes. This conclusion can explain the two experimental phenomena about human P-gp in some extent. These studies have provided a detailed exploration into human P-gp rearrangement process and given an energy insight into the TMD reorientation during P-gp transition.     

Inhibition of multidrug resistance-linked P-glycoprotein (ABCB1) function by 5'-fluorosulfonylbenzoyl 5'-adenosine: evidence for an ATP analogue that interacts with both drug-substrate-and nucleotide-binding sites

5'-Fluorosulfonylbenzonyl 5'-adenosine (FSBA) is an ATP analogue that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases, and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC(50 )= 0.21 mM) and the binding of 8-azido[α-(32)P]ATP (IC(50) = 0.68 mM). In addition, (14)C-FSBA cross-links to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photo-cross-linking of P-gp with [(125)I]iodoarylazidoprazosin (IAAP; IC(50) = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA cross-links to residues within or nearby the NBDs but not in the transmembrane domains, and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analogue that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated cross-linking is observed only at the NBDs.     

The translocation mechanism of P-glycoprotein

Multidrug transporters are involved in mediating the failure of chemotherapy in treating several serious diseases. The archetypal multidrug transporter P-glycoprotein (P-gp) confers resistance to a large number of chemically and functionally unrelated anti-cancer drugs by mediating efflux from cancer cells. The ability to efflux such a large number of drugs remains a biological enigma and the lack of mechanistic understanding of the translocation pathway used by P-gp prevents rational design of compounds to inhibit its function. The translocation pathway is critically dependent on ATP hydrolysis and drug interaction with P-gp is possible at one of a multitude of allosterically linked binding sites. However, aspects such as coupling stoichiometry, molecular properties of binding sites and the nature of conformational changes remain unresolved or the centre of considerable controversy. The present review attempts to utilise the available data to generate a detailed sequence of events in the translocation pathway for this dexterous protein.     

How conformational changes can affect catalysis,inhibition and drug resistance of enzymes with induced-fit binding mechanism such as the HIV-1 protease

A central question is how the conformational changes of proteins affect their function and the inhibition of this function by drug molecules. Many enzymes change from an open to a closed conformation upon binding of substrate or inhibitor molecules. These conformational changes have been suggested to follow an induced-fit mechanism in which the molecules first bind in the open conformation in those cases where binding in the closed conformation appears to be sterically obstructed such as for the HIV-1 protease. In this article, we present a general model for the catalysis and inhibition of enzymes with induced-fit binding mechanism. We derive general expressions that specify how the overall catalytic rate of the enzymes depends on the rates for binding, for the conformational changes, and for the chemical reaction. Based on these expressions, we analyze the effect of mutations that mainly shift the conformational equilibrium on catalysis and inhibition. If the overall catalytic rate is limited by product unbinding, we find that mutations that destabilize the closed conformation relative to the open conformation increase the catalytic rate in the presence of inhibitors by a factor exp(ΔΔG_C/RT) where ΔΔG_C is the mutation-induced shift of the free-energy difference between the conformations. This increase in the catalytic rate due to changes in the conformational equilibrium is independent of the inhibitor molecule and, thus, may help to understand how non-active-site mutations can contribute to the multi-drug-resistance that has been observed for the HIV-1 protease. A comparison to experimental data for the non-active-site mutation L90M of the HIV-1 protease indicates that the mutation slightly destabilizes the closed conformation of the enzyme. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Inhibition of yeast mitochondrial F1-ATPase, F0F1-ATPase and submitochondrial particles by rhodamines and ethidium bromide

ATP hydrolysis by F1-ATPase is strongly inhibited by cationic rhodamines; neutral rhodamines are very poor inhibitors. Rhodamine 6G is a noncompetitive inhibitor of purified F0F1-ATPase and submitochondrial particles, however, an uncompetitive inhibitor of F1-ATPase (KI approximately equal to 2.4 microM for all three enzyme forms). Ethidium bromide is a noncompetitive inhibitor of F0F1-ATPase, submitochondrial particles and also F1-ATPase (KI approximately equal to 270 microM). Neither of the inhibitors affects the negative cooperativity (nH approximately equal to 0.7). The non-identical binding sites for rhodamine 6G and ethidium bromide are located on the F1-moiety and are topologically distinct from the catalytic site. Binding of the inhibitors prevents the conformational changes essential for energy transduction. It is concluded that the inhibitor binding sites are involved in proton translocation. In F1-ATPase, binding of MgATP at a catalytic site causes conformational changes, which allosterically induce the correct structure of the rhodamine 6G binding site. In F0F1-ATPase, this conformation of the F1-moiety exists a priori, due to allosteric interactions with F0-subunits. The binding site for ethidium bromide on F1-ATPase does not require substrate binding at the catalytic site and is not affected by F0F1-subunit interactions.     

Targeted molecular dynamics simulation studies of binding and conformational changes in E. coli MurD

Enzymes involved in the biosynthesis of bacterial peptidoglycan, an essential cell wall polymer unique to prokaryotic cells, represent a highly interesting target for antibacterial drug design. Structural studies of E. coli MurD, a three-domain ATP hydrolysis driven muramyl ligase revealed two inactive open conformations of the enzyme with a distinct C-terminal domain position. It was hypothesized that the rigid body rotation of this domain brings the enzyme to its closed active conformation, a structure, which was also determined experimentally. Targeted molecular dynamics 1 ns-length simulations were performed in order to examine the substrate binding process and gain insight into structural changes in the enzyme that occur during the conformational transitions into the active conformation. The key interactions essential for the conformational transitions and substrate binding were identified. The results of such studies provide an important step toward more powerful exploitation of experimental protein structures in structure-based inhibitor design.     

Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein

ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.     

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.     

Comparative structural dynamics of Tyrosyl-tRNA synthetase complexed with different substrates explored by molecular dynamics

Several molecular dynamics simulations of S. aureus Tyrosyl-tRNA synthetase (TyrRS) in its free form and complexed with Tyr, ATP, tyrosyl adenylate and inhibitor respectively have been carried out to investigate the ligand-linked conformational stability changes associated with its catalytic cycle. The results show that unliganded S. aureus TyrRS samples a more relaxed conformational space than substrate-bound TyrRS. There are three high flexibility regions encompassing residues 114–118, 128–133, and 226–238 respectively. The region which includes the KMSKS motif (KFGKS in S. aureus TyrRS) shows the highest difference in fluctuations. Hydrogen bond network formed by Tyr, ATP, tyrosyl adenylate and inhibitor with S. aureus TyrRS is discussed. Our simulations suggest the induced-fit conformational changes of the KMSKS loop as follows: the KMSKS loop of substrate-free S. aureus TyrRS adopts an open conformation. The tyrosine binds in the pocket with the KMSKS loop balancing between semi-open and open forms. The ATP binding induces the KMSKS loop to the open form. After the Tyr-AMP is formed, the first two residues of KMSKS loop twists in an anticlockwise direction and drives the loop in a conformation similar to the closed one, while those of the last three GKS residues adopt a conformation between semi-open and open conformation. This conformational change may probably be involved in the initial tRNA binding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular modeling study for conformational changes of Sirtuin 2 due to substrate and inhibitor binding

Sirtuin is a member of NAD(+)-dependent deacetylase family. The structural details of Sirtuin 2 (SIRT2) complex will be very useful to discover the drug which might have beneficial effects on various diseases like cancer, diabetes, etc. Unfortunately, SIRT2 complex structure is not available yet, hence molecular docking was carried out to dock the substrate (NAD(+) and acetylated lysine) and inhibitor (sirtinol) in the NAD(+) binding site. The suitable binding orientation of substrate and inhibitor in the SIRT2 active site was selected and subjected to 5 ns molecular dynamics simulations to adjust the binding orientation of inhibitor and substrate as well as to identify the conformational changes in the active site. The result provides an insight about 3D SIRT2 structural details as well as the importance of F96 in deacetylation function. In addition, our simulations revealed the displacement of F96 upon substrate and inhibitor binding, inducing an extended conformation of loop3 and changing its interactions with the rest of SIRT2. We believe that our study could be helpful to gain a structural insight of SIRT2 and to design the receptor-based inhibitors.     

The Interactions of P-Glycoprotein with Antimalarial Drugs,Including Substrate Affinity,Inhibition and Regulation

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10⁻⁶ cm/sec, followed by amodiaquine around 20 x 10⁻⁶ cm/sec; both mefloquine and artesunate were around 10 x 10⁻⁶ cm/sec. Methylene blue was between 2 and 6 x 10⁻⁶ cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.