Drug-binding Domain Research Articles

Interactions of human P-glycoprotein transport substrates and inhibitors at the drug binding domain: Functional and molecular docking analyses

Rhodamine 123 (R123) transport substrate sensitizes P-glycoprotein (P-gp) to inhibition by compound 2c (cis–cis) N,N-bis(cyclohexanolamine)aryl ester isomer in a concentration-dependent manner in human MDR1-gene transfected mouse T-lymphoma L5178 cells as shown previously. By contrast, epirubicin (EPI) concentration changes left unaltered 2c IC50 values of EPI efflux. To clarify this discrepancy, defined molecular docking (DMD) analyses of 12 N,N-bis(cyclohexanolamine)aryl esters, the highly flexible aryl ester analog 4, and several P-gp substrate/non-substrate inhibitors were performed on human P-gp drug- or nucleotide-binding domains (DBD or NBD). DMD measurements yielded lowest binding energy (LBE, kcal/mol) values (mean±SD) ranging from −11.8±0.54 (valspodar) to −3.98±0.01 (4). Lys234, Ser952 and Tyr953 residues formed H-bonds with most of the compounds. Only 2c docked also at ATP binding site (LBE value of −6.9±0.30kcal/mol). Inhibition of P-gp-mediated R123 efflux by 12 N,N-bis(cyclohexanolamine)aryl esters and 4 significantly correlated with LBE values. DMD analysis of EPI, 3H-1EPI, 3H-2EPI, 14C-1EPI, 14C-2EPI, R123 and 2c before and after previous docking of each of them indicated that pre-docking of either 2c or EPI significantly reduced LBE of both EPI and R123, and that of both 3H-2EPI and 14C-2EPI, respectively. Since the clusters of DBD amino acid residues interacting with EPI were different, if EPI docked alone or after pre-docking of EPI or 2c, the existence of alternative secondary binding site for EPI on P-gp is credible. In conclusion, 2c may allocate the drug-binding pocket and reduce strong binding of EPI and R123 in agreement with P-gp inhibition experiments, where 2c reduced efflux of EPI and R123.

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli.

The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions--Y49S, V127A, V127G, D153E, and G288C--mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions--F453C and L486W--were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

Multiple Drug Transport Pathways through Human P-Glycoprotein.

P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Combinatorial In Silico Drug‐Lead Optimization for Targeted Inhibitors of P‐Glycoprotein

P‐glycoprotein (P‐gp) is an ATP‐powered efflux pump of the plasma membrane that is responsible for removing toxins from cells. While this is beneficial in normal cells, P‐gp overexpression is often responsible for the failure of chemotherapies in cancer and HIV treatments. Because P‐gp transports drugs that encompass a wide variety of structures, multidrug resistances (MDR) to many unrelated therapeutics is often a consequence of P‐gp overexpression. Attempts to identify inhibitors of P‐gp that could be used to reverse MDR have often failed because they are also pumped out of the cell. We recently identified three inhibitors that target the nucleotide binding domains of P‐gp and avoid the drug binding domains. These compounds reverse MDR in cancer cell lines. A big problem in optimizing any drug‐lead is that the number of possible chemical variations of the lead always out‐numbers those that can be synthesized and tested. We present here a novel computational approach that generates many thousands of compounds related to a drug‐lead structure, analyzes them for interaction with the target protein, and rates them for improvements in target binding potential. We have applied this approach to inhibitors of P‐gp that reverse multidrug resistances in cancer and report on the application of these variants to high performance computational drug docking experiments with Pgp. These methods aimed at expediting the optimization of drug leads should be generalizable to many different systems. Supported by NIH NIGMS [Grant 1R15‐GM094771‐01A1] to PDV and JGW, and SMU Undergraduate Research Assistantship, SMU Engaged Learning, and Hamilton Undergraduate Research Scholarships to KO.

Transport of Alzheimer's Associated Amyloid‐β Through Membranes Catalyzed by P‐Glycoprotein

The broad range of substrates that can be transported by ABCB1 plasma membrane transporters has been well established. Of these transporters, MDR1 P‐glycoprotein (P‐gp), has been extensively studied and is best known for the abilities to confer general multidrug resistance to populations of cancer cells and for preventing pharmaceutical products from crossing the blood brain barrier (BBB). P‐gp action has recently been also implicated in Alzheimer's disease (AD). One of the clearance pathways of amyloid‐β, thought to be a causative agent in neurodegeneration and symptom onset in AD, is suggested to be through P‐gp across the BBB. In this study, a P‐gp model with amyloid‐β bound to drug binding domains was used in targeted molecular dynamics simulation (TMD) of the catalytic cycle. TMD employed four different crystallographic conformations to move the protein through open inside to open outside conformations. Transport of amyloid‐β through the membrane was observed during the putative catalytic cycles. Results suggest that the catalytic cycle resulted in movement of amyloid‐β to P‐gp structures productive for release to the extracellular space. These simulations have elucidated specific molecular interactions between P‐gp and amyloid‐β that result in amyloid‐β transport. Such studies have enabled a detailed and targeted biochemical analysis of amyloid‐β transport through the BBB. This work is supported by NIH NIGMS [Grant 1R15‐GM094771‐01A1] to PDV and JGW, SMU Research Council, SMU Dean's Research Council, the SMU Center for Drug Discovery, Design and Delivery, and the Communities Foundation of Texas, Dallas.

In silico screening for inhibitors of p-glycoprotein that target the nucleotide binding domains.

Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp.

Inhibition of energy transduction in P‐glycoprotein (974.3)

Cancer chemotherapy failures often involve the over‐expression of ABC‐transporters like the MDR1 P‐glycoprotein (P‐gp). These transporters normally protect the cell by pumping toxins and xenobiotics across the plasma membrane. Selection for cells over‐expressing P‐gp during chemotherapy, however, results in populations that are drug resistant. Recently, we used massively parallel in silico drug docking to identify molecules predicted to interact with higher affinity at the energy transducing structures of P‐gp than at the drug transporting structures. In vitro assays verified that the compounds inhibited verapamil‐stimulated ATP hydrolysis and only marginally stimulated basal ATP hydrolysis activities. This indicates that these compounds do not interact with drug binding domains. The effects of the identified compounds on ATP binding was then studied using spin‐labeled ATP analogs and ESR spectroscopy. Results showed that some of the identified compounds decreased ATP binding stoichiometry to P‐gp while others decreased ATP binding affinities. These studies further showed that inhibition by the compounds was reversible. The investigations suggest that the discovered P‐gp inhibitors may make good drug leads. Experiments using ESR spectroscopy and site‐specific spin‐labeling of the drug binding domain are currently underway that may shed further light on the molecular mechanism of inhibition.Grant Funding Source: NIGMS1R15GM094771 ‐ 01A1, Communities Foundations of Texas

Dynamic elucidation of drug transport pathways in human P‐glycoprotein (974.5)

Chemotherapy resistant, recurrent cancers most commonly express members of the ABCB1 class of plasma membrane transporters, specifically MDR1 P‐glycoprotein transporter (P‐gp). At high levels of expression, the P‐gp transporter lowers the intracellular concentrations of chemotherapeutics to below effective therapeutic levels. Because P‐gp can transport a broad range of substrates, such expression leads to general multidrug resistance in the population of cancerous cells. Dynamic P‐gp models which transition through putative catalytic cycles have been previously created by us using targeted molecular dynamics simulations (TMD) with four different crystallographic conformations of related proteins. In the present study, these simulations have been expanded to include transport substrates docked into different initial sites within the drug binding domain of P‐gp. Even though drug movement in these simulations was not targeted by TMD, transport of chemotherapeutics (daunorubicin and doxorubicin) and a P‐gp transport substrate (verapamil) through the membrane by human P‐gp was observed during the putative catalytic cycles. Initial results suggest that different starting positions of transport substrate in the “open to the cytoplasm” conformation resolve into a common transport pathway through the membrane as P‐gp closes to the cytoplasm and opens to the extracellular space.Grant Funding Source: NIH 1R15GM094771 ‐ 01A1, Communities Foundations of TX

Localization and Substrate Selectivity of Sea Urchin Multidrug (MDR) Efflux Transporters

In this study, we cloned, expressed and functionally characterized Stronglycentrotus purpuratus (Sp) ATP-binding cassette (ABC) transporters. This screen identified three multidrug resistance (MDR) transporters with functional homology to the major types of MDR transporters found in humans. When overexpressed in embryos, the apical transporters Sp-ABCB1a, ABCB4a, and ABCG2a can account for as much as 87% of the observed efflux activity, providing a robust assay for their substrate selectivity. Using this assay, we found that sea urchin MDR transporters export canonical MDR susbtrates such as calcein-AM, bodipy-verapamil, bodipy-vinblastine, and mitoxantrone. In addition, we characterized the impact of nonconservative substitutions in the primary sequences of drug binding domains of sea urchin versus murine ABCB1 by mutation of Sp-ABCB1a and treatment of embryos with stereoisomeric cyclic peptide inhibitors (QZ59 compounds). The results indicated that two substitutions in transmembrane helix 6 reverse stereoselectivity of Sp-ABCB1a for QZ59 enantiomers compared with mouse ABCB1a. This suggests that subtle changes in the primary sequence of transporter drug binding domains could fine-tune substrate specificity through evolution.

Fibroblast Growth Factor Receptor 2 Homodimerization Rapidly Reduces Transcription of the Pluripotency Gene Nanog without Dissociation of Activating Transcription Factors

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.

Structural characterization of the PPIase domain of FKBP51, a cochaperone of human Hsp90

Steroid hormone receptors are key components of mammalian stress and sex hormone systems. Many of them rely on the Hsp90 chaperone system for full function and are further fine-tuned by Hsp90-associated peptidyl-prolyl isomerases such as FK506-binding proteins 51 and 52. FK506-binding protein 51 (FKBP51) has been shown to reduce glucocorticoid receptor signalling and has been genetically associated with human stress resilience and with numerous psychiatric disorders. The peptidyl-prolyl isomerase domain of FKBP51 contains a high-affinity binding site for the natural products FK506 and rapamycin and has further been shown to convey most of the inhibitory activity on the glucocorticoid receptor. FKBP51 has therefore become a prime new target for the treatment of stress-related affective disorders that could be amenable to structure-based drug design. Here, a series of high-resolution structures of the peptidyl-prolyl isomerase domain of FKBP51 as well as a cocrystal structure with the prototypic ligand FK506 are described. These structures provide a detailed picture of the drug-binding domain of FKBP51 and the molecular binding mode of its ligand as a starting point for the rational design of improved inhibitors.

Recognition of Sulfonylurea Receptor (ABCC8/9) Ligands by the Multidrug Resistance Transporter P-glycoprotein (ABCB1): FUNCTIONAL SIMILARITIES BASED ON COMMON STRUCTURAL FEATURES BETWEEN TWO MULTISPECIFIC ABC PROTEINS

ATP-sensitive K(+) (K(ATP)) channels are the target of a number of pharmacological agents, blockers like hypoglycemic sulfonylureas and openers like the hypotensive cromakalim and diazoxide. These agents act on the channel regulatory subunit, the sulfonylurea receptor (SUR), which is an ABC protein with homologies to P-glycoprotein (P-gp). P-gp is a multidrug transporter expressed in tumor cells and in some healthy tissues. Because these two ABC proteins both exhibit multispecific recognition properties, we have tested whether SUR ligands could be substrates of P-gp. Interaction with P-gp was assayed by monitoring ATPase activity of P-gp-enriched vesicles. The blockers glibenclamide, tolbutamide, and meglitinide increased ATPase activity, with a rank order of potencies that correlated with their capacity to block K(ATP) channels. P-gp ATPase activity was also increased by the openers SR47063 (a cromakalim analog), P1075 (a pinacidil analog), and diazoxide. Thus, these molecules bind to P-gp (although with lower affinities than for SUR) and are possibly transported by P-gp. Competition experiments among these molecules as well as with typical P-gp substrates revealed a structural similarity between drug binding domains in the two proteins. To rationalize the observed data, we addressed the molecular features of these proteins and compared structural models, computerized by homology from the recently solved structures of murine P-gp and bacterial ABC transporters MsbA and Sav1866. Considering the various residues experimentally assigned to be involved in drug binding, we uncovered several hot spots, which organized spatially in two main binding domains, selective for SR47063 and for glibenclamide, in matching regions of both P-gp and SUR.

Mutations Define Cross-talk between the N-terminal Nucleotide-binding Domain and Transmembrane Helix-2 of the Yeast Multidrug Transporter Pdr5

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K(m)((ATP)) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.

Therapeutic Strategies for Long-QT Syndrome

The discovery of genetic defects underlying long-QT syndrome (LQTS) has allowed to identify important genotype-phenotype correlations that are now being used for risk stratification. The next challenge is to exploit the new information on the pathophysiology of the disease derived from molecular genetics to devise more effective therapies. The successful response of LQT1 patients to β-blockers, the QT-shortening action of sodium channel blockers in at least some LQT3 patients, and the importance of maintaining adequate plasma potassium levels in LQT2 patients clearly demonstrate the importance of selecting therapy in the context of the molecular substrate. The hurdles on the road toward the development of novel therapeutic strategies are represented by the variable expressivity of the disease, the high prevalence of “private” mutations, and the role of genetic and nongenetic factors that act as modifiers of the phenotype. Given the large number of mutations identified and their phenotypic complexity, it is clearly impossible to anticipate a scenario in which each mutation will be managed through a specific therapy. However, the evidence that mutations in the LQTS gene may be clustered based on their electrophysiological profile and on their response to specific drugs may provide the rationale for the development of mutation-specific therapies. In this article, we will review the most relevant genotype-phenotype features of LQTS and the strategies explored to develop novel therapeutic approaches. ### Definition and Prevalence The congenital LQTS is an inherited arrhythmogenic disease characterized by abnormally prolonged QT interval leading to life-threatening arrhythmias in the presence of a structurally normal heart.1 Different clinical variants of LQTS have been identified. Romano Ward syndrome, transmitted as an autosomal dominant trait, is the most common form of LQTS and presents only a cardiac phenotype. Less prevalent variants of LQTS, such as Andersen and Timothy syndromes or the recessive Jervell and Lange-Nielsen syndromes, also present …

Novel Lamivudine Resistance

With great interest I have read and appreciate the recent publication by Warner and coworkers (6). Those authors described the effects of a novel mutation in the hepatitis B virus (HBV) polymerase reverse transcriptase domain, namely, the rtL80I substitution that confers resistance to lamivudine. This mutation was initially described to occur in Japanese patients with lamivudine treatment failure, but its impact remained to be investigated (3). Not surprisingly, based on molecular modeling of the reverse transcriptase domain of the HBV polymerase, Warner et al. (6) concluded that this novel mutation is not located in the supposed drug binding domain. Nevertheless, the fact that rtL80I is able to mediate lamivudine resistance also in vitro, as shown by phenotyping, is of high general interest and deserves foremost attention for two reasons. First, the fact that mutations in the periphery of the nucleos(t)ide binding site may lead to resistance has to be taken into account in all future resistance testing approaches. Second, the observation by Warner and colleagues (6) may be true not only for lamivudine but also for other drugs and may thus have an impact also on further therapy and monitoring regimens, mainly as the therapy for chronic HBV infection is becoming more and more complex (1). Especially for adefovir, it is believed that besides mutations, host factors such as failures in liver uptake, defective or nonappropriate esterases, and phosphorylation failures may also confer resistance to the drug (5). Yet, although extensively discussed, novel mutations (e.g., see references 2 and 4) or mutations in the periphery of the nucleos(t)ide binding site have been taken into account only marginally or even disbelieved (5). Based on the observations of Warner et al., investigators should systematically check as to whether new or unexpected mutation patterns in the nucleos(t)ide binding site in concert with or even without mutations in the periphery lead to resistance or predispose to resistance to antivirals.

Probing lipid- and drug-binding domains with fluorescent dyes

A series of 2- and 3-OH Nile red dyes was prepared in order to generate water-soluble probes that could be used to probe lipid binding to proteins. Various substitutions in positions 2-/3-, 6-, and 7-shifted wavelengths while maintaining the environmental sensitivity of Nile red. In order to increase the solubility of the dyes in aqueous solutions, we attached butyric acid groups to the 2- or 3-OH position. In addition, phenothiazine dyes, which exhibited particularly long excitation properties, were synthesized and tested for the first time. All dyes showed Stoke’s shifts of 70–100 nm and changes in excitation and emission of over 100 nm, depending on the hydrophobicity of the environment. Binding studies with bovine serum albumin and the non-specific lipid transfer protein SCP2 revealed emission changes of more than 30 nm upon binding to the protein and a five-fold increase in emission intensity. Titration of the dye-loaded proteins with various lipids or drugs replaced the dye and thereby reversed the shift in wavelength intensity. This allowed us to estimate the lipid binding affinity of the investigated proteins. For SCP2, isothermal calorimetry (ITC) data verified the titration experiments. NMR titration experiments of SCP2 with Nile red 2- O-butyric acid ( 1a) revealed that the dye is bound within the lipid binding pocket and competes with lipid ligands for this binding site. These results give valuable insight into lipid and drug transport by proteins outside and inside cells.

Selective control of endothelial cell proliferation with a synthetic dimerizer of FGF receptor-1

Basic fibroblast growth factor (bFGF) is a potent angiogenic molecule, but its therapeutic use is limited by mitogenic effects on multiple cell types. To specifically activate FGF signaling in endothelial cells, a chimeric FGF receptor was generated that contained a modified FK506 drug-binding domain (F36V) fused to the FGF receptor-1 (FGFR1) cytoplasmic domain. Human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells were retrovirally transduced with this chimeric receptor, and the effects of administering synthetic receptor-dimerizing ligands were studied. As expected, both control and transduced cells proliferated in response to bFGF treatment; however, only transduced endothelial cells exhibited dose-dependent proliferative responses to dimerizer treatment. Dimerizer-induced proliferation was MEK-dependent and was accompanied by MAP kinase phosphorylation, indicating that the chimeric receptor utilizes signaling pathways similar to endogenous FGFR1. Although bFGF stimulated wound re-epithelialization in HUVECs (which natively express FGFR1 and FGFR4), chemical dimerization of FGFR1 did not; this suggests FGFR4 may control migration in these cells. The ability to selectively activate receptor subtypes should facilitate the study of signaling pathways in vitro and in vivo beyond what can be accomplished with nonselective natural ligands, and it may eventually permit stimulation of graft cell angiogenesis without driving overgrowth of host cells.

Drug‐induced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine

Fluoxetine (Prozac) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro. We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K+ and SCN5A Na+ channels heterologously expressed in HEK293 cells. Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration-dependent, block of hERG current (IhERG). Biochemical studies showed that both compounds also caused concentration-dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of IhERG but did not alter fluoxetine's effect on hERG channel protein trafficking. Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce IhERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug-induced long QT syndrome.

Recent Advances in the New Generation Taxane Anticancer Agents

AbstractFor Abstract see ChemInform Abstract in Full Text.