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Structures of the influenza A virus M2 proton channel have been determined by X-ray crystallography in the open conformation, and by NMR in the closed state. Whereas the X-ray structure shows a single inhibitor molecule in the middle of the channel, four inhibitor molecules bind the channel’s outer surface in the NMR structure. Although in both structures the strongest hot spots (i.e., regions which substantially contribute to the free energy of binding any potential ligand) lie inside the pore, hot spots also are found at exterior locations. By considering all available models, we propose the primary drug binding site is inside the pore, but that exterior binding also occurs under appropriate conditions.

The pore site in microtubules has been studied with the use of Hexaflutax, a fluorescent probe derived from paclitaxel. The compound is active in cells with similar effects to paclitaxel, indicating that the pore may be a target to microtubule stabilizing agents. While other taxanes bind microtubules in a monophasic way, thus indicating a single type of sites, Hexaflutax association is biphasic. Analysis of the phases indicates that two different binding sites are detected, reflecting two different modes of binding, which could arise from different arrangements of the taxane or fluorescein moieties in the pore. Association of the 4-4-20 antifluorescein monoclonal antibody-Hexaflutax complex to microtubules remains biphasic, thus indicating that the two phases observed arise from two different poses of the taxane moiety., This work was supported in part by grant BIO2007-61336 from the Ministry of Science and Innovation to J.F.D., BIPPED-CM from Comunidad de Madrid to J.F.D. and J.M.A., and grant MOST No. 2006DFA31490 to W.S.F.

The role of the ATP-binding cassette ABCB1 in mediating the resistance to chemotherapy in many forms of cancer has been well established. The protein is also endogenously expressed in numerous barrier and excretory tissues, thereby regulating or impacting on drug pharmacokinetic profiles. Given these prominent roles in health and disease, a great deal of biochemical, structural and pharmacological research has been directed towards modulating its activity. Despite the effort, only a small handful of compounds have reached the later stages of clinical trials. What is responsible for this poor return on the heavy research investment? Perhaps the most significant factor is the lack of information on the location, physical features and chemical properties of the drug-binding site(s) in ABCB1. This minireview outlines the various strategies and outcomes of research efforts to pin-point the sites of interaction. The data may be assimilated into two working hypotheses to describe drug binding to ABCB1; (a) the central cavity and the (b) domain interface models.

The mechanism of interaction of hypoglycemic drugs, glimepiride and glipizide with human serum albumin (HSA) has been studied using fluorescence spectroscopy. The results are discussed in terms of the binding parameters, thermodynamics of the binding process, nature of forces involved in the interaction, identification of drug binding site on serum albumin and the fluorescence quenching mechanism involved. The association constants were of the order of 105 and glipizide was found to have much higher affinity for HSA than glimepiride at all temperatures. Thermodynamic parameters for the binding suggested that hydrophobic interactions are primarily involved in the binding of these drugs to HSA. However, glimepiride and glipizide appear to cause temperature-dependent conformational changes in the albumin molecule and, therefore, the nature of interaction varied with temperature. Glimepiride and glipizide bind to both site I and site II on HSA, but the primary interaction occurs at site II. The binding region in site II is different for the two drugs. Stern-Volmer analysis of quenching data indicated that tryptophan residues of HSA are not fully accessible to the drugs and a predominantly dynamic quenching mechanism is involved in the binding. Results can provide useful insight into prediction of competitive displacement of these drugs by other co-administered drugs and excipients, resulting in serious fluctuations of the blood glucose levels in diabetic patients.

Human serum albumin (HSA) is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants, Such binding plays a crucial role in determining the ADME (absorption, distribution, metabolism, and excretion) and bioavailability of the pollutants. The binding interaction between HSA and acetic acid (C2), octanoic acid (C8) and dodecanoic acid (C 12) has been investigated by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. For the study of the fatty acid interaction with the two drug-binding sites on HSA, two fluorescent probes, dansylamide and dansyl-L-proline were employed in the displacement measurements. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocattalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations from these three approaches were very similar. HSA did not show any change in the fluorescence and electrochemical experiments after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential mode, indicating an independent and non-cooperative binding. The calculated association constant and binding ratio were 3.1 x 10(6) L/mol and I with drug binding Site I, 1.1 X 10(7) L/mol and I with Site II, and 7.0x 10(4) L/mol and 4 with the tryptophin site, respectively. The measurements with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of the C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.

Mitoxantrone (MTX) is a clinically used antitumor anthracycline, which is made available to the target tissues by transport protein human serum albumin (HSA). Being less toxic unlike other member of this family, its binding characteristics are therefore of immense interest. The circular dichroism (CD), fluorescence and Fourier transform infrared (FTIR) spectroscopies were employed to elucidate the mode and the mechanism for this interaction. MTX binding is characterized by one high affinity binding site with the association constants of the order of 10 5 . Correlation between stability of N-MTX (drug bound N form of HSA) and B-MTX (drug bound B form of HSA) complexes with drug distribution has been discussed. The molecular distance, r , between donor (HSA) and acceptor (MTX) was estimated according to Forster’s theory of non-radiation energy transfer. The features of MTX induced structural perturbation of human serum albumin (HSA) has been studied in detail by CD and FTIR analysis. Domain I was assigned to possess high affinity binding site for MTX. Molecular docking showed that the MTX binds HSA to a non-classical drug binding site. The binding dynamics was expounded by synchronous fluorescence, thermodynamic parameters and molecular modeling, which entails that hydrophobic interactions, hydrogen bonding and electrostatic forces, stabilizes the interaction.

Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.

Influenza A and B viruses contain proton-selective ion channels, A/M2 and BM2, respectively, and the A/M2 channel activity is inhibited by the drugs amantadine and its methyl derivative rimantadine. The structure of the pore-transmembrane domain has been determined by both x-ray crystallography [Stouffer et al. (2008) Nature 451:596–599] and by NMR methods [Schnell and Chou (2008) Nature 451:591–595]. Whereas the crystal structure indicates a single amantadine molecule in the pore of the channel, the NMR data show four rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40–45 with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electrophysiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24–36 into BM2, yielded a pH-activated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.

ABCG2 confers resistance to cancer cells by mediating the ATP-dependent outward efflux of chemotherapeutic compounds. Recent studies have indicated that the protein contains a number of interconnected drug binding sites. The present investigation examines the coupling of drug binding to ATP hydrolysis. Initial drug binding to the protein requires a high-affinity interaction with the drug binding site, followed by transition and reorientation to the low-affinity state to enable dissociation at the extracellular face. [3H]Daunomycin binding to the ABCG2R482G isoform was examined in the nucleotide-bound and post-hydrolytic conformations. Binding of [3H]daunomycin was displaced by ATP analogues, indicating transition to a low-affinity conformation prior to hydrolysis. The low-affinity state was observed to be retained immediately post-hydrolysis. Therefore, the dissociation of phosphate and/or ADP is likely to be responsible for resetting of the transporter. The data indicate that, like ABCB1 and ABCC1, the ‘power stroke’ for translocation in ABCG2R482G is the binding of nucleotide.

Binding modalities of doxorubicin (DOX), a widely used antineoplastic anthracyline antibiotic with hemoglobin (Hb) have been studied. The protein and the ligand were prepared using CORINA and protonated with insight II. The best conformation was sought by employing GOLDV. Molecular modeling calculations showed that DOX binds Hb to a non-classical drug binding site. The alpha subunit of Hb has been assigned to posses the binding site for DOX with a binding affinity (Ka) = 16.98 x10 3 mol -1 . The interaction was found to be thermodynamically favorable (ΔG° = -66.23 KJmol -1 ). The analysis of DOX binding site to Hb suggested that the types of interactions that contribute in this binding are hydrophobic contacts, hydrogen bonding and electrostatic interactions. Keywords: hemoglobin; doxorubicin; molecular docking; binding site Background: It is well established that free molecules of drug can act at the target site. This is because free drugs are rapidly distributed into tissues, which facilitate direct binding to tissue proteins. The binding of drugs to plasma and tissue proteins is common. This is considered to be an important factor to determine the pharmacokinetics and pharmacodynamics of drugs. The formation of drug–protein adducts can cause cellular and tissue toxicity which may be either intrinsic or idiosyncratic in nature

EmrE is a Small Multidrug Resistance transporter (SMR) family member that mediates counter transport of protons and hydrophobic cationic drugs such as tetraphenylphosphonium (TPP+), ethidium, propidium and dequalinium. It is thought that the selectivity of the drug binding site in EmrE is defined by two negatively charged glutamate residues within a hydrophobic pocket formed from six of the α-helices, three from each monomer of the asymmetric EmrE homodimer. It is not apparent how such a binding pocket accommodates drugs of various sizes and shapes or whether the conformational changes that occur upon drug binding are identical for drugs of diverse chemical nature. Here, using electron cryomicroscopy of EmrE two-dimensional crystals we have determined projection structures of EmrE bound to three structurally different planar drugs, ethidium, propidium and dequalinium. Using image analysis and rigorous comparisons between these density maps and the density maps of the ligand-free and TPP+-bound forms of EmrE, we identify regions within the transporter that adapt differentially depending on the type of ligand bound. We show that all three planar drugs bind at the same pocket within the protein as TPP+. Furthermore, our analysis indicates that, while retaining the overall fold of the protein, binding of the planar drugs is accompanied by small rearrangements of the transmembrane domains that are different to those that occur when TPP+ binds. The regions in the EmrE dimer that are remodelled surround the drug binding site and include transmembrane domains from both monomers.

We disclose the first selective, nonpeptidic, small-molecule somatostatin receptor subtype 5 (SST5R) antagonists that were identified by a chemogenomics approach based on the analysis of the homology of amino acids defining the putative consensus drug binding site of SST5R. With this strategy, opioid, histamine, dopamine, and serotonine receptors were identified as the closest neighbors of SST5R. The H1 antagonist astemizole was chosen as a seed structure and subsequently transformed into a SST5 receptor antagonist with nanomolar binding affinity devoid of the original target activity.

Human alpha(1)-acid glycoprotein (AGP) is an important drug binding plasma protein which affects pharmacokinetical properties of various therapeutic agents. For the first time, interpretation of the induced circular dichroism (ICD) spectra of drug-AGP complexes is presented yielding valuable information on the protein binding environment. ICD spectra were obtained by novel ligands of which AGP induced optical activity have never been reported (primaquine, mefloquine, propranolol, terazosin, carbamazepine, rhodamine B) and by re-investigation of ICD spectra of protein-bound drugs published earlier (chlorpromazine, dipyridamole, prazosin). Spectroscopic features of the ICD and absorption bands of drugs combined with native AGP indicated chiral non-degenerate exciton coupling between the guest chromophore and the indole ring of an adjacent tryptophan (Trp) residue. Results of additional CD experiments performed by using recombinant AGP mutants showed no changes in the ligand binding ability of W122A in sharp contrast with the W25A which was unable to induce extrinsic CD signal with either ligand. Thus, these findings unequivocally prove that, likely via pi-pi stacking mechanism, Trp25 is essentially involved in the AGP binding of drugs studied here as well as of related compounds. Survey of the AGP binding data published in the literature support this conclusion. Our results provide a fast and efficient spectroscopic tool to determine the inclusion of ligand molecules into the beta-barrel cavity of AGP where the conserved Trp25 is located and might be useful in ligand-binding studies of other lipocalin proteins.

The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.

Marine ladder-shaped polyether toxins are implicated in neurological symptoms of fish-borne food poisonings. The toxin gambierol, produced by the marine dinoflagellate Gambierdiscus toxicus, belongs to the group of ladder-shaped polyether toxins and inhibits Kv3.1 channels with nanomolar affinity through a mechanism of gating modification. Binding determinants for gambierol localize at the lipid-exposed interface of the pore forming S5 and S6 segments, suggesting that gambierol binds outside of the permeation pathway. To explore a possible involvement of the voltage-sensing domain (VSD), we made different chimeric channels between Kv3.1 and Kv2.1, exchanging distinct parts of the gating machinery. Our results showed that neither the electro-mechanical coupling nor the S1-S3a region of the VSD affect gambierol sensitivity. In contrast, the S3b-S4 part of the VSD (paddle motif) decreased gambierol sensitivity in Kv3.1 more than 100-fold. Structure determination by homology modeling indicated that the position of the S3b-S4 paddle and its primary structure defines the shape and∖or the accessibility of the binding site for gambierol, explaining the observed differences in gambierol affinity between the channel chimeras. Furthermore, these findings explain the observed difference in gambierol affinity for the closed and open channel configurations of Kv3.1, opening new possibilities for exploring the VSDs as selectivity determinants in drug design.

The major drug binding site of sodium channels is inaccessible from the extracellular side, drug molecules can only access it either from the membrane phase, or from the intracellular aqueous phase. For this reason, ligand-membrane interactions are as important determinants of inhibitor properties, as ligand-protein interactions. One-way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane. In this electrophysiology study we used three different pH values: 6.0, 7.3, and 8.6 to test the significance of the protonation-deprotonation equilibrium in drug access and affinity. We investigated drugs of several different indications: carbamazepine, lamotrigine, phenytoin, lidocaine, bupivacaine, mexiletine, flecainide, ranolazine, riluzole, memantine, ritanserin, tolperisone, silperisone, ambroxol, haloperidol, chlorpromazine, clozapine, fluoxetine, sertraline, paroxetine, amitriptyline, imipramine, desipramine, maprotiline, nisoxetine, mianserin, mirtazapine, venlafaxine, nefazodone, and trazodone. We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics. As expected, we observed a strong correlation between the acidic dissociation constant (pKa) of drugs and the pH-dependence of their potency. Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation. Our data are best explained by a model where drug molecules can be trapped in at least two chemically different environments: A hydrophilic trap (which may be the aqueous cavity within the inner vestibule), which favors polar and less lipophilic compounds, and a lipophilic trap (which may be the membrane phase itself, and/or lipophilic binding sites on the channel). Rescue from the hydrophilic and lipophilic traps can be promoted by alkalic and acidic extracellular pH, respectively.

Fluorescence emission and anisotropy are widely used to measure the binding parameters and kinetic behavior of reactions that cause a change in the rotational time of a fluorescent molecule. We report here fluorescence emission and anisotropy behavior of a newly synthesized novel naphthalene base fluorophore (methyl 3-[(6-{[2-(tert-butoxy)-2-oxoethyl] (4-methoxyphenyl)amino}naphthalen-2-yl)formamido]propanoate) in several solution conditions including its binding to human and bovine serum albumin proteins both in their native and denatured states. The fluorescence yield of the compound substantially increased inside hydrophobic protein surface and ~30 nm decrease in Stokes’ shift, compared to aqueous solution, was observed. Shift in fluorescence excitation peak position from the absorption peak of the molecule was ~8 nm in protein solution. This indicated possible alteration of excited state geometry of the compound by the globular fold of albumins. In addition, we measured the steady state fluorescence anisotropy of the molecule to evaluate several thermodynamic parameters and the results suggested the binding was energetically favorable. The measured ΔG° was ~−30 kJ mol−1 and the derived dissociation constant was ~10−6 M. The molecular docking analysis further highlighted the nonspecific association of the compound with the proteins and hydrophobic forces may have a significant role in the binding processes. Under the denatured condition of the protein, the compound lost its binding efficacy and reduction in fluorescence intensity was observed. Thus, the molecule appears as a new fluorescence probe to report the nature of its binding site in terms of increased fluorescence quantum yield and decreased Stokes’ shift. It can also report the changes in the binding site due to global change in protein structure such as unfolding/misfolding often linked to several human disorder. Further it could be useful to detect and study the drug binding site of specific protein of interest. Electronic supplementary material The online version of this article (doi:10.1186/s40064-015-1333-8) contains supplementary material, which is available to authorized users.

AS-3201 [(3R)-2'-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4'(1'H)-pyrrolo[1,2-a]pyrazine]-1',2,3',5(2'H)-tetrone] is a structurally novel and stereospecifically potent aldose reductase (AKR1B; EC 1.1.1.21) inhibitor, which contains a succinimide ring that undergoes ring-opening at physiological pH levels. To delineate intermolecular interactions governing its favorable pharmacokinetic profile, the interaction of AS-3201 (R-isomer) with plasma proteins, especially human serum albumin (HSA), was examined in comparison with that of the optical antipode (S-isomer). Fluorescence, kinetic, and high-performance frontal analyses showed that the R-isomer is more strongly bound than the S-isomer to sites I and II on HSA, and the R-isomer is particularly protected from hydrolysis, suggesting that the stable HSA-R-isomer complex contributes to its prolonged activity. The thermodynamic parameters for the specific binding indicated that in addition to hydrophobic interactions, hydrogen bonds contribute significantly to the R-isomer complex formation. (13)C NMR observations of the succinimide ring (5-(13)C enriched), which are sensitive to its ionization state, suggested the presence of a hydrogen bond between the R-isomer and HSA, and (19)F NMR of the pendent benzyl ring (2-(19)F) evaluated the equilibrium exchange dynamics between the specific sites. Furthermore, fatty acid binding or glycation (both are site II-oriented perturbations) inhibited the binding to one of the specific sites and reduced the stereospecificity of HSA toward the isomers, although the clinical influence of these perturbations on the R-isomer binding ratio seemed to be minor. Thus, the difference in the interaction mode at site II might be a major cause of the stereospecificity; this is discussed on the basis of putative binding modes. The present results, together with preliminary absorption and distribution profiles, provide valuable information on the stereospecific pharmacokinetic and pharmacodynamic properties of the R-isomer relevant for the therapeutic treatment of diabetic complications.

Paclitaxel resistance mutations in Chinese hamster ovary cells frequently alter a cluster of leucine residues in the H6-H7 loop region of beta-tubulin. To gain further insight into the role of this region in microtubule assembly and drug resistance, site-directed mutagenesis was used to systematically change amino acid L215. The mutated genes were cloned into a tetracycline-regulated expression vector and transfected into wild-type cells. Most of the mutations destabilized microtubule assembly, causing a decreased fraction of tubulin to appear in the microtubule cytoskeleton. In each case, the decreased level of assembly was associated with paclitaxel resistance and increased colcemid sensitivity. In two cases, however, the alteration did not significantly perturb the level of assembled tubulin or confer resistance to paclitaxel. One of these, L215V, produced little or no detectable phenotype, while the other, L215I, conferred increased sensitivity to paclitaxel. The increased drug sensitivity did not extend to epothilone A, a drug that binds to the same site and has a mechanism of action similar to that of paclitaxel, or colcemid, a drug with an opposing mechanism of action and a distinct binding site. Moreover, L215I conferred enhanced paclitaxel sensitivity at very low levels of expression, and sensitivity was not further enhanced in cells with higher levels of expression, implying that paclitaxel acts substoichiometrically. These properties, along with the proximity of L215 to the drug binding site, suggests that the L215I substitution may enhance the binding or effectiveness of paclitaxel. Our studies confirm the importance of the H6-H7 loop of beta-tubulin in microtubule assembly and resistance to antimitotic drugs. They also identify the first mammalian mutation shown to specifically increase sensitivity to paclitaxel.

Two-layered and three-layered ONIOM calculations were performed to compare the binding energies of 8-Cl TIBO inhibitor when bound into the human immunodeficiency virus reverse transcriptase binding pocket and a Y181C variant. Both consisted of 20 residues within a radius of 15 A. A combination of different methods [MP2/6-31G(d), B3LYP/6-31G(d,p), and PM3] were performed to take advantage of ONIOM's layering strategy analysis. The obtained results clearly indicate that the Y181C mutation reduces the binding affinity and stability of the inhibitor by approximately 8-9 kcal/mol as obtained from different combined MO:MO methods. Analyses regarding the energetic components of the interaction and deformation energies for 8-Cl TIBO inhibitor upon binding were also examined extensively. Additional calculations involving the interaction energies between 8-Cl TIBO with individual residues surrounding the binding pocket were performed at MP2/6-31G(d,p) and B3LYP/6-31G(d,p) levels of theory to gain more insight into the energetic differences of wild-type and Y181C mutant type at the atomistic level.

Daunomycin (DM) is a clinically used antitumor anthracycline antibiotic, which is transported primarily by human serum albumin (HSA) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of DM. A new optical biosensor technique based on the resonant mirror was used to characterize interaction of DM with HSA at different temperatures and the affinity constants were obtained. The HSA-DM interaction is exothermic with having favorable enthalpy and entropy followed by the integrated van't Hoff equation analysis. Fluorescence studies showed that DM has an ability to quench the intrinsic fluorescence of HSA through a static quenching procedure according to the Stern-Volmer equation and DM displays a pH-dependent binding affinity to HSA. Molecular modeling calculations showed that the DM binds HSA to a non-classical drug binding site and further analysis of the binding site of DM within the HSA molecule suggested that hydrophobic contacts, hydrogen bond formation and electrostatic interactions account for the binding of DM.

Kv subunits are accessory proteins that modify gating of Kv1 channels. Kv1.3 subunits bind to the N termini of Kv1.5 -subunits and induce fast N-type inactivation, slow the rate of deactivation, and alter the voltage dependence and kinetics of channel activation. The N terminus of a Kv subunit and quaternary ammonium compounds bind to the inner pore of Kv1 channels; however, it is unknown to what extent the pore binding sites for drugs and Kv subunits overlap. Here, we used site-directed Ala mutagenesis to scan residues of the Kv1.5 pore to define the binding site for Kv1.3 subunits. Individual mutations of five residues in the S6 domain (Val505, Ile508, Leu510, Val512, and Val516) greatly retarded or prevented Kv1.3 induced inactivation, and reduced effects on Kv1.5 deactivation. Mutation of Thr479 and Thr480 enhanced Kv1.3-induced N-type inactivation. None of the Ala mutations prevented the Kv1.3-induced negative shifts in the voltage dependence of activation or slow C-type inactivation, suggesting that these gating effects are mediated by an interaction other than the one for N-type inactivation. Thr479, Thr480, Val505, Ile508, and Val512, of Kv1.5 channels are also important interaction sites for the anthranilic acid S0100176 (Nbenzyl-N-pyridin-3-ylmethyl-2-(toluene-4-sulfonylamino)benzamide hydrochloride). Leu510 and V516A prevented Kv1.3-induced inactivation but did not alter drug block. Block of Kv1.5 by S0100176 was reduced and voltage-dependent in the presence of Kv1.3 but not in the presence of an Ntruncated form of the Kv subunit. Thus, residues in the pore of Kv1.5 required for N-type inactivation overlap with but are not identical to the drug binding site. Voltage gated K (Kv) channels have diverse cellular functions, including maintenance of the resting potential, repolarization of action potentials, indirect modulation of neurohormone release, volume regulation, and K transport. Kv channels are formed by coassembly of four poreforming -subunits. The diversity of Kv channel structure and function is enhanced by heteromultimerization of different -subunits (Coetzee et al., 1999) or by coassembly with small accessory Kv subunits (Rhodes et al., 1997). Kv subunits can bind to Kv -subunits to form 44 complexes that modify the biophysical properties and/or expression levels of the heteromultimeric channel. For ex

Summary The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant. Comparative investigation of E. coli with and without a plasmid-encoded Cfr showed a decreased drug binding to ribosomes in the presence of Cfr. As chloramphenicol/florfenicol and clindamycin have partly overlapping drug binding sites on the ribosome, the most likely explanation is that Cfr modifies the RNA in the drug binding site. This hypothesis was supported by drug footprinting data that showed both a decreased drug binding and an enhanced reverse transcriptase stop at position 2504, which corresponds to a modification at position A2503 at the drug binding site. A 45 n long RNA fragment containing the appropriate region was isolated and MALDI-TOF mass spectrometry in combination with tandem mass spectrometry showed an additional methylation at position A2503. Moreover, reduced methylation was detected at nucleotide C2498. The results show that Cfr is an RNA methyltransferase that targets nucleotide A2503 and inhibits ribose methylation at nucleotide C2498, thereby causing resistance to chloramphenicol, florfenicol and clindamycin.

The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C 4 (LTC 4 ) is one of the highest affinity substrates of MRP1. In this study, we have synthesizedand characterized a novel photoreactive azido analogue of LTC 4 (AALTC 4 ). The specificity of AALTC 4 binding to MRP1 was confirmed using an LTC 4 -specific monoclonal antibody. Moreover, binding with radioiodinated [ 1 2 5 I]AALTC 4 (or IAALTC 4 ) to MRP1 was dramatically competed with unmodified LTC 4 and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC 4 binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC 4 binding to MRP1. Using AALTC 4 together with a panel of epitope-specific and LTC 4 -specific monoclonal antibodies, we identified LTC 4 binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPrl, QCRL1, and MRPm6) showed LTC 4 binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC 4 , indicating that MSD0 contains an LTC 4 binding site. Higher resolution mapping of additional LTC 4 binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC 4 and digested with trypsin to isolate specific regions of MRP1 that interact with LTC 4 . These results confirmed that sequences in MSD0 interact with IAALTC 4 . Other regions that were photoaffinity labeled by IAALTC 4 include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC 4 binding domains in MRP1 and provide the first direct evidence for LTC 4 binding within MSD0.

Blockade of hERG K(+) channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the two-dimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-pi interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltage-gated K(+) channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site.

Progesterone and the antiprogestin RU38486 have been reported as non-transported modulators of P-glycoprotein-mediated drug efflux. However, their hormonal properties limit their potential for clinical trials. The present work shows that some derivatives from either progesterone/RU38486 or estradiol, displaying differential interaction with hormone receptors, bind to P-glycoprotein and chemosensitize the growth of MDR1-transfected cells to vinblastine more strongly than does RU38486. Structure comparison of the compounds indicates that the highly hydrophobic estradiol derivative RU49953, which does not interact with any hormone receptor, inhibits P-glycoprotein-mediated drug efflux very efficiently, as monitored by flow cytometry, and prevents drug site photoaffinity labeling by azidopine. It induces a much higher chemosensitization than the well-known P-glycoprotein modulator verapamil, which is itself more efficient than RU38486. RU49953 therefore constitutes a promising new lead for steroid-type modulators of multidrug resistance.

Agosterol A (AG-A) is a novel agent that reverses P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1)-meditated multidrug resistance (MDR). We have synthesized [125I]11-azidophenyl agosterol A (azidoAG-A), a photoaffinity analog of AG-A, and characterized its binding to P-gp in membrane vesicles prepared from multidrug-resistant P-gp-overexpressing KB-C2 cells. The photoanalog photolabeled intact P-gp and both the N- and C-terminal fragments of P-gp. [125I]AzidoAG-A is transported by P-gp and the intracellular accumulation of both [125I]azidoAG-A and [3H]AG-A in KB-C2 cells was lower than that in the parental drug-sensitive KB-3-1 cells. [125I]AzidoAG-A bound to the drug binding site(s) on P-gp because photoaffinity labeling of P-gp was inhibited by a variety of known P-gp substrates, including anticancer, reversing, and anti-human immunodeficiency virus (HIV) agents. The binding of [125I]azidoAG-A to P-gp differs from the binding of other photolabeled probes such as iodoaryl-azidoprazosin (IAAP) to P-gp and from the binding of [125I]azidoAG-A to MRP1 based on the differing effects of flupentixol and glutathione (GSH) on their binding. Thus, [125I]azidoAG-A will be a useful tool to elucidate the structure and function of P-gp because it directly binds to the drug binding site(s) on P-gp, is transported by P-gp, and exhibits different P-gp binding characteristics than IAAP.

Summary Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A 2A adenosine receptor (A 2A AR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone 15 N– 1 H NMR signals in A 2A AR were individually assigned. These NMR probes provided insight into the role of Asp52 2.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 246 6.48 , and delineated the structural response to variable efficacy of bound drugs across A 2A AR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp52 2.50 .

The druggability of a target is defined by the likelihood of a certain target binding site to be amendable to functional modulation by a small molecule in vivo. Thus, druggability depends on the ability of the developed small molecule to reach the target site, the properties of the ligand binding pocket and our ability to develop chemical matter that efficiently interact with the drug binding site of interest. Historically enzymes have been the main drug targets because the inhibition of their activity can be easily assayed and catalytic centres are often attractive drug binding sites. However, despite considerable effort, a number of classical enzyme families have not been successfully targeted. More recently protein-protein interactions received considerable attention and several clinical inhibitors have now been developed. Despite the considerable progress made expanding target space, a large number of targets with a very strong rationale for targeting remain intractable. In the following chapter I will summarize progress made in developing inhibitors for challenging drug binding sites and emerging target families.

V-PYRRO/NO [O(2)-vinyl-1-(pyrrolidin-1-yl) diazen-1-ium-1,2-diolate] and V-PROLI/NO (O2-vinyl-[2-(carboxylato) pyrrolidin-1-yl]diazen-1-ium-1,2-diolate), two structurally similar diazeniumdiolate derivatives, were designed as liver-selective prodrugs that are metabolized by cytochrome P450 isoenzymes, with subsequent release of nitric oxide (NO). Yet, their efficacy in the treatment of nonalcoholic fatty liver disease (NAFLD) and their comparative pharmacokinetic and metabolic profiles have not been characterized. The aim of the present work was to compare the effects of V-PYRRO/NO and V-PROLI/NO on liver steatosis, glucose tolerance, and liver fatty acid composition in C57BL/6J mice fed a high-fat diet, as well as to comprehensively characterize the ADME (absorption, distribution, metabolism and excretion) profiles of both NO donors. Despite their similar structure, V-PYRRO/NO and V-PROLI/NO showed differences in pharmacological efficacy in the murine model of NAFLD. V-PYRRO/NO, but not V-PROLI/NO, attenuated liver steatosis, improved glucose tolerance, and favorably modified fatty acid composition in the liver. Both compounds were characterized by rapid absorption following i.p. administration, rapid elimination from the body, and incomplete bioavailability. However, V-PYRRO/NO was eliminated mainly by the liver, whereas V-PROLI/NO was excreted mostly in unchanged form by the kidney. V-PYRRO/NO was metabolized by CYP2E1, CYP2C9, CYP1A2, and CYP3A4, whereas V-PROLI/NO was metabolized mainly by CYP1A2. Importantly, V-PYRRO/NO was a better NO releaser in vivo and in the isolated, perfused liver than V-PROLI/NO, an effect compatible with the superior antisteatotic activity of V-PYRRO/NO. In conclusion, V-PYRRO/NO displayed a pronounced antisteatotic effect associated with liver-targeted NO release, whereas V-PROLI/NO showed low effectiveness, was not taken up by the liver, and was eliminated mostly in unchanged form by the kidney.

The interaction of the novel disaccharide anthracycline MEN 10755 with human serum albumin (HSA) was investigated by visible absorption and fluorescence spectroscopies and by ultrafiltration. Notably, MEN 10755 binds serum albumin far stronger than doxorubicin. Albumin binding results into a drastic quenching of the intrinsic fluorescence of MEN 10755; a binding constant of 1.1×105 was determined from fluorescence data. To localize the HSA binding site of MEN 10755 competition experiments were carried out with ligands that are selective for the different drug binding sites of the protein. No relevant competition effects were seen in the case of warfarin, diazepam and hemin, known ligands of sites I, II and III, respectively. Modest effects were observed following addition of palmitic acid that targets the several fatty acid binding sites of the protein. In contrast, extensive displacement of the bound anthracycline was achieved upon addition of ethacrinic acid. On the basis of these results, it is proposed that MEN 10755 binds serum albumin tightly to a non-canonical surface binding site for which it competes specifically with ethacrinic acid.

Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

Accepted manuscript version. Published version at http://doi.org/10.1111/febs.13200. During the last decades antimicrobial resistance has become a global health problem. Metallo-β-lactamases (MBLs) which are broad-spectrum β-lactamases that inactivate virtually all β-lactams including carbapenems, are contributing to this health problem. In this study a novel MBL variant, termed VIM-26, identified in a Klebsiella pneumoniae isolate was studied. VIM-26 belongs to the Verona integron-encoded metallo-β-lactamase (VIM) family of MBLs and is a His224Leu variant of the well-characterized VIM-1 variant. In this study, we report the kinetic parameters, minimum inhibitory concentrations and crystal structures of a recombinant VIM-26 protein, and compare them to previously published data on VIM-1, VIM-2 and VIM-7. The kinetic parameters and minimum inhibitory concentration determinations show that VIM-26, like VIM-7, has higher penicillinase activity but lower cephalosporinase activity than VIM-1 and VIM-2. The four determined VIM-26 crystal structures revealed mono- and di-zinc forms, where the Zn1 ion has distorted tetrahedral coordination geometry with an additional water molecule (W2) at a distance of 2.6–3.7 Å, which could be important during catalysis. The R2 drug binding site in VIM-26 is more open compared to VIM-2 and VIM-7 and neutrally charged due to Leu224 and Ser228. Thus, the VIM-26 drug binding properties are different from the VIM-2 (Tyr224/Arg228) and VIM-7 (His224/Arg228) structures, indicating a role of these residues in the substrate specificity.

Human topoisomerase 1B controls the topological state of supercoiled DNA allowing the progression of fundamental cellular processes. The enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by cleaving one DNA strand and forming a transient protein-DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its interaction with the DNA substrate, likely affecting the strand rotation and religation rate. The mutation also causes a slight rearrangement of the active site and of the drug binding site, providing an additional explanation for the lowered effect of camptothecin toward the mutant.

Present study reveals that the subdomain IIA cavity of two homologous serum albumins (HSA, BSA) has inherent mutual structural and functional deviations which render noticeable difference in behavior toward specific ligands. The major drug binding site (subdomain IIA) of HSA is found to be largely hydrophobic while that of BSA is partially exposed to water. Larger shift in REE spectra and greater change in solvent reorganization energy of coumarin 343 (C343)-anion in HSA clearly reveals that binding pocket is relatively large and water molecules penetrate deeper into it unlike BSA. The individual response of proteins to perturbation by ligands is found to be way different. Although the subdomain IIA is primarily anion receptive (prefers anionic ligands), the present study suggests that HSA may also like to bind neutral guests due to its remarkable conformational features. Actually, HSA is capable of adopting favorable conformation like mechanical slide-wrench, when required, to accommodate neutral ligands [e.g., coumarin 314 (C314)], as well. But due to less flexible solution structure, BSA behaves like fixed mechanical spanners and hence is not very responsive to C314. Therefore, the generally speaking functional-structural similarities of homologous proteins can be apparent and needs to be analyzed exhaustively.

Background: Inhibiting PfDHODH kills malaria parasites, but the potential for drug resistance is unknown. Results: Selections gave several categories of resistance mutations. Several mutants were hypersensitive to other drugs. Conclusion: Resistance to PfDHODH inhibitors is largely though mutations in or amplification of the target gene, PfDHODH. Significance: Resistance to PfDHODH inhibitors is possible but often increases sensitivity to other compounds., Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.

P-glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad-specificity, ATP-dependent efflux transporter of diverse lipophilic neutral or cationic compounds. Previously, we identified two positively cooperative drug-binding sites of P-glycoprotein involved in transport [Shapiro, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 130-137]. The H site is selective for Hoechst 33342 and colchicine. The R site is selective for rhodamine 123 and anthracyclines. Substrate binding to one site stimulates transport by the other. In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123. Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner. In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually. Non-P-glycoprotein-specific effects of prazosin on membrane fluidity and permeability were excluded. These results indicate the existence of a third drug-binding site on P-glycoprotein with a positive allosteric effect on drug transport by the H and R sites. This allosteric site appears to be one of the sites of photoaffinity labeling of P-glycoprotein by [125I]iodoarylazidoprazosin [Safa, A. R., Agresti, M., Bryk, D. & Tamai, I. (1994) Biochemistry 33, 256-265] and is likely not to be capable of drug transport.

The elevated expression of ATP binding cassette (ABC) multidrug transporters in multidrug-resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms. Multidrug transporters interact directly with the drug substrates. This review summarizes current insights into the mechanism(s) by which ATP hydrolysis is coupled to drug transport in bacterial LmrA and its human homolog P-glycoprotein. In addition, the relevance of these insights for other ABC transporters will be discussed, (C) 2001 Editions scientifiques et medicales Elsevier SAS.

The P-glycoprotein multidrug transporter is a 170-kDa efflux pump which exports a diverse group of natural products, chemotherapeutic drugs, and hydrophobic peptides across the plasma membrane, driven by ATP hydrolysis. The transporter has been proposed to interact with its drug substrates within the membrane environment; however, much remains to be learned about the nature and number of the drug binding site(s). The two nucleotide binding domains are responsible for ATP binding and hydrolysis, which is coupled to drug movement across the membrane. In recent years, P-glycoprotein has been purified and functionally reconstituted in amounts large enough to allow biophysical studies. The use of spectroscopic techniques has led to insights into both its secondary and tertiary structure, and its interaction with nucleotides and drugs. In this review, we will summarise what has been learned by application to purified P-glycoprotein of fluorescence spectroscopy, circular dichroism spectroscopy and infra-red spectroscopy.

We investigated the action of the phenylalkylamines verapamil and N-methyl-verapamil on the Kv1.3 potassium channel using the whole-cell configuration of the patch-clamp technique. Our goal was to identify their binding as a prerequisite for using the phenylalkylamines as small, well-defined molecular probes, not only to expand the structural findings made with peptide toxins or by crystallization, but also to use them as lead compounds for the generation of more potent and therefore more specific K+ channel modulators. Competition experiments with charybdotoxin, known to interact with external residues of Kv1.3, showed no interaction with verapamil. The internal application of quarternary N-methyl-verapamil in combination with verapamil suggested competition for the same internal binding site. Verapamil affinity was decreased 6 fold by a mutation (M395V) in a region of the internal pore which forms part of the internal tetraethylammonium (TEA+) binding site, although mutations at neighbouring residues (T396 and T397) were without effect. Modification of C-type inactivation by mutations in the internal pore suggest that this region participates in the inactivation process. The action of phenylalkylamines and local anaesthetics on L-type Ca2+ channels and Na+ channels, respectively, and verapamil on Kv1.3 indicate very similar blocking mechanisms. This might allow the use of these compounds as molecular probes to map the internal vestibule of all three channel types. British Journal of Pharmacology (1999) 127, 1065–1074; doi:10.1038/sj.bjp.0702599

Quinobenzoxazine A-62176, developed from the antibacterial fluoroquinolones, is active in vitro andin vivo against murine and human tumors. It has been previously claimed that A-62176 is a catalytic inhibitor of mammalian topoisomerase II that does not stabilize the cleaved complex. However, at low drug concentrations and pH 6–7, we have found that A-62176 can enhance the formation of the cleaved complex at certain sites. Using a photocleavage assay, mismatched sequences, and competition experiments between psorospermin and A-62176, we pinpointed the drug binding site on the DNA base pairs between positions +1 and +2 relative to the cleaved phosphodiester bonds. A 2:2 quinobenzoxazine-Mg2+self-assembly model was previously proposed, in which one drug molecule intercalates into the DNA helix and the second drug molecule is externally bound, held to the first molecule and DNA by two Mg2+ bridges. The results of competition experiments between psorospermin and A-62176, as well as between psorospermin and A-62176 and norfloxacin, are consistent with this model and provide the first evidence that this 2:2 quinobenzoxazine-Mg2+ complex is assembled in the presence of topoisomerase II. These results also have parallel implications for the mode of binding of the quinolone antibiotics to the bacterial gyrase-DNA complex.

The soil bacterium Streptomyces peucetius produces two widely used anticancer antibiotics, doxorubicin and daunorubicin. Present within the biosynthesis gene cluster in S. peucetius is the drrAB operon, which codes for a dedicated ABC (ATP binding cassette)-type transporter for the export of these two closely related antibiotics. Because of its dedicated nature, the DrrAB system is believed to belong to the category of single-drug transporters. However, whether it also contains specificity for other known substrates of multidrug transporters has never been tested. In this study we demonstrate under both in vivo and in vitro conditions that the DrrAB system can transport not only doxorubicin but is also able to export two most commonly studied MDR substrates, Hoechst 33342 and ethidium bromide. Moreover, we demonstrate that many other substrates (including verapamil, vinblastine, and rifampicin) of the well studied multidrug transporters inhibit DrrAB-mediated Dox transport with high efficiency, indicating that they are also substrates of the DrrAB pump. Kinetic studies show that inhibition of doxorubicin transport by Hoechst 33342 and rifampicin occurs by a competitive mechanism, whereas verapamil inhibits transport by a non-competitive mechanism, thus suggesting the possibility of more than one drug binding site in the DrrAB system. This is the first in-depth study of a drug resistance system from a producer organism, and it shows that a dedicated efflux system like DrrAB contains specificity for multiple drugs. The significance of these findings in evolution of poly-specificity in drug resistance systems is discussed.

Isonicotinic acid hydrazide (isoniazid or INH) is a front line antitubercular pro-drug that is converted to its active form, isonicotinyl-NAD, by the bacterial catalase-peroxidase KatG. Understanding the role of KatG in the INH activation process has been hampered by a lack of knowledge of the actual drug binding site. In this work, we have investigated the binding of INH in the main access channel of KatG with a combination of molecular dynamics, using an enhanced-sampling technique (metadynamics), X-ray crystallography, and site-directed mutagenesis. The metadynamics simulations show that there are several weak drug binding sites along the access channel. Moreover, the simulations evidence that complete entrance to the heme active site is impeded by an aspartate residue (D141) located above the heme. This has been confirmed by structural and functional analysis of the D141A mutant, leading to the first X-ray crystallography evidence of INH at the heme access channel. © 2014 American Chemical Society., Financial support was provided by MINECO (Grants CTQ2011-25871 and BFU2012-36827), GENCAT (Grant 2009SGR-1309), NSERC (Discovery Grant 9600), and the Canada Research Chair Program (to P.C.L.)

We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.

Pgp is an atypical translocating ATPase, with low affinity for ATP and high constitutive ATPase activity. Pgp also has an unusually broad specificity for hydrophobic substrates, including many chemotherapeutic drugs. Transport studies in reconstituted systems indicate that drug transport requires ATP hydrolysis and is active, generating a drug concentration gradient. Binding of drugs and ATP to Pgp induces conformational changes in the protein, and the drug binding site is conformationally coupled to the NBDs. Evidence accumulated to date suggests that the transporter interacts directly with nonpolar substrates within the membrane environment, and may act as a drug flippase, moving drugs from the inner to the outer leaflet of the bilayer. Chemosensitizers that block the action of Pgp are proposed to act as alternative substrates, but their high rate of spontaneous flip-flop across the membrane results in futile cycling of the transporter.

18 pages, 7 figures, 3 tables., Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block., This study was supported by FIS 95/0318 (Dr Valenzuela), CICYT SAF96–0042 (Dr Tamargo), and National Institute of Health grants HL-47599 (Dr Snyders), HL-46681 (Drs Snyders and Tamkun), and HL-49330 (Dr Tamkun).

1 The interaction of the indolizin sulfone SR33557 with the multidrug resistance P-glycoprotein (P-gp), was used to explore the nature of drug binding site(s) on this transporter. The steady-state accumulation of [ 3 H]-vinblastine in P-gp expressing CH r B30 cells was increased by SR33557 with greater potency than verapamil. Furthermore, SR33557 potentiated the aAnity of verapamil to modulate vinblastine transport when added simultaneously. 2 Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CH r B30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations. However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity. This is indicative of a non-competitive interaction, most likely at distinct sites. 3 The specific [ 3 H]-vinblastine binding to P-gp in CH r B30 cell membranes was displaced by SR33557 with an IC50 of 8.3+4.5 nM. Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P-gp indicating a negative allosteric eAect on the vinca alkaloid acceptor site. 4 These results demonstrate that SR33557 interacts with a site on P-gp which is distinct from, but allosterically linked to the vinca alkaloid site. The apparent broad substrate specificity displayed by P-gp may be explained by a multiple drug binding site model.

Despite considerable therapeutic success with the antimalarial 4-aminoquinolines such as chloroquine, there is serious doubt about the future of this drug class due mainly to the development and spread of parasite resistance throughout endemic areas. In this article we review the possible biochemical and molecular basis of resistance. Based on our current understanding we have considered the possibility of developing strategies which may allow the aminoquinolines to once again be used effectively against P. falciparum. Our conclusions are that drug resistance is the result of a reduced rate of drug uptake which in turn reduces the amount of drug available to bind the target. The basis for this reduced accumulation could be an altered pH gradient making the food vacuole more alkaline or the parasite cytosol more acidic, an efflux pump removing drug directly from the membrane or any other process which will reduce the rate of drug uptake. Central to the effectiveness of this resistance mechanism is the transient availability of a high affinity, low capacity drug binding site (possibly haem) within the parasite. Resistance reversers such as verapamil influence the apparent Ka for this drug binding phenomenon via an increased drug uptake rate. We demonstrate that by chemical modification of the aminoquinolines, producing predictable alterations in their physicochemical properties, that it is possible to minimise the verapamil sensitive component of resistance and reduce significantly cross-resistance patterns without loss in absolute activity. Based on these views we suggest that the aminoquinoline antimalarials still have a role to play in the cheap, safe and effective chemotherapy of falciparum malaria.

Recombinant viruses of echovirus 12 carrying mutations of a rhodanine-resistant or -dependent variant, were investigated, and five single mutations each inducing a rhodanine-resistant or -dependent phenotype were defined. Four mutations are localized in the capsid protein VP1, and the fifth exchange is in VP4. All original and recombinant viruses were shown to be stabilized by the antiviral drug rhodanine against heat inactivation. Hence, resistant and dependent variants still seem able to bind rhodanine, and apparently none of the exchanges affects the putative drug binding site. We hypothesize that drug resistance and dependence are consequences of an increased flexibility of the virus capsid.