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Papers by Anna Seelig

Bioorganic & Medicinal Chemistry Letters, 2006

7-Deoxypaclitaxel, 10-deacetoxypaclitaxel and 10-deacetoxy-7-deoxypaclitaxel were prepared and ev... more 7-Deoxypaclitaxel, 10-deacetoxypaclitaxel and 10-deacetoxy-7-deoxypaclitaxel were prepared and evaluated for their ability to promote assembly of tubulin into microtubules, their cytotoxicity against NCI/ADR-RES cells and for their interactions with Pglycoprotein in bovine brain microvessel endothelial cells. The three compounds were essentially equivalent to paclitaxel in cytotoxicity against NCI/ADR-RES cells. They also appeared to interact with P-glycoprotein in the endothelial cells with the two 10-deacetoxy compounds having less interaction than paclitaxel and 7-deoxypaclitaxel.

Protein unfolding is a dynamic cooperative process with many short-lived intermediates. Cooperati... more Protein unfolding is a dynamic cooperative process with many short-lived intermediates. Cooperativity means that similar molecular elements act dependently on each other. The thermodynamics of protein unfolding can be determined with differential scanning calorimetry (DSC). The measurement of the heat capacity provides the temperature profiles of enthalpy, entropy and free energy. The thermodynamics of protein unfolding is completely determined with these thermodynamic properties. We emphasise the model-independent analysis of the heat capacity. The temperature profiles of enthalpy H(T), entropy S(T) and free energy G(T) can be obtained directly by a numerical integration of Cp(T). In evaluating different models for protein unfolding. It is essential to simulate all thermodynamic properties, not only the heat capacity. A chemical equilibrium two-state model is a widely used approximation to protein unfolding. The model assumes a chemical equilibrium between only two protein conforma...

Methods and Principles in Medicinal Chemistry, 2004

Seminars in Cancer Biology, 1997

We review how P-glycoprotein recognizes a wide variety of compounds and how it carries its substr... more We review how P-glycoprotein recognizes a wide variety of compounds and how it carries its substrates across membranes. Amino acid substitutions that affect the substrate specificity of P-glycoprotein have been found scattered throughout the molecule. In particular, some amino acid () residues in the putative transmembrane domain TM 1 together with TM5᎐6 and TM11᎐12 may help to govern substrate specificity. The features that substrates for P-glycoprotein share are also discussed. The amphipathy of a substrate may decide whether the substrate can be intercalated into the lipid bilayer of the membrane. In addition, only certain molecular volumes and tertiary structures may make it possible for the substrate to fit into the substrate-() binding site s of P-glycoprotein.

Bioorganic & Medicinal Chemistry Letters, 2006

A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core tha... more A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core that reduces interaction with P-glycoprotein in bovine brain microvessel endothelial cells is described. Modification and derivatization of the C10 po\sition were carried out using a substrate controlled hydride addition to a key C9 and C10 diketone intermediate. The analogs were tested for tubulin assembly and cytotoxicity, and were shown to retain potency similar to paclitaxel. P-glycoprotein interaction was examined using a rhodamine assay and it was found that simple hydrolysis or epimerization of the C10 acetate of paclitaxel and Taxol C can reduce interaction with the P-glycoprotein transporter that may allow for increased permeation of taxanes into the brain.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990

The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gin-Phe-Phe-Gly-Leu-Met amide) ... more The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gin-Phe-Phe-Gly-Leu-Met amide) and three of its antagonists were derived by measuring the Gibbs adsorption isotherm, providing information on the surface activity, the molecular shape, and the pK values of the different molecules. The following three antagonists were investigated: [

Protein stability is of utmost importance in many areas of life sciences and life science technol... more Protein stability is of utmost importance in many areas of life sciences and life science technology. The gold standard for assessing protein stability is the measurement of the heat capacity Cp(T), showing large peaks at the temperature of cold and heat unfolding. We first demonstrate that important thermodynamic properties, including the free energy, can be obtained from Cp(T) temperature profiles without the necessity of an unfolding model. This is illustrated with experimental examples taken from the literature. We then compare all thermodynamic results with chemical and statistical unfolding models. An often-cited chemical equilibrium two-state model makes partially incorrect predictions. New models are introduced, which are in excellent agreement with the experimental results. We then show that the free energy is not a good criterion to judge protein stability. More useful parameters are discussed, including protein cooperativity. The new parameters are embedded in a well-defi...

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987

The bindln~ of the local anesthetic dilmcaine to myers composed of 1-paimitoyl-2-uleoyl-sn-giyeer... more The bindln~ of the local anesthetic dilmcaine to myers composed of 1-paimitoyl-2-uleoyl-sn-giyeero-3plmsplmclmline was studied with a Langmuir trough at pH 5.5 (22°C, 0.1 M NaO). At this pH value only the charged form of the local anesthetic exists in solution. Charged dibucaine was found to be surface active and to penetrate into the lipid monolayer, with the hydrophobic part of the molecule being accommodated between the fatty acyl chains of the Hpid. The dibucaine intercalation could be quantitated by meastwing the expansion of the film area, AA, at constant surface pressme, yr. At a given surface pressure, AA increased with increasing dilmcaine in the buffer phase. On the other hand, keeping the dibucaine concentration constant, the area increase, AA, was strongly dependent on the surface pressure. The area increase, AA, was large at low surface pressure and decreased with increasing sm~ace pressure. A plot of the relative change in surface area, /tA/A, versus the surface pressure yielded straight lines in the pressure range of 25-36 mN/m for five different concentrations. The AA/A vs. vr isotherms intersected at vr-39.5 + 1 mN/m with AA =0, indicating that charged dilmcaine apparentiy can no longer penetrate into the monolayer film. By making judicial assumptions about the area requirement of dilmcaine the monolayer expansion curves could be transformed into true binding isotherms. Dibucaine binding isotherms were constructed for different monolayer pressures and were compared to a bilayer binding isotherm measured under similar conditions with ultraviolet spectroscopy. The best agreement between monolayer and bilayer binding data was obtained for a monolayer held at a pressure of 30.7 to 32.5 raN/m, which can thus be considered as the bilayer-monolayer equivalence lW&,sure. It is further suggested from this analogy that the binding of dibueaine does not change the internal pressure in the bilayer phase, at least not in the concentration range of physiological interest (0-2 mM dilmcaiue) but induces a lateral expansion. At higher molar ratios of cationic dilmeaine to lipid, Xb, in the monolayer (x b >0.20) the area increase is larger than would be expected from the molecular dimensions of dibueaine. This is probably due to charge repulsion effects, which at still higher molar ratios (x b > 0.6) lead to a micemsation. The pressure dependence of the intercalation of cationic dibueaine into lipid membranes may also be of relevance for the phenomenon of pressure reversal in anesthesia.

SSRN Electronic Journal, 2021

Protein unfolding is a dynamic cooperative equilibrium between short lived protein conformations.... more Protein unfolding is a dynamic cooperative equilibrium between short lived protein conformations. The Zimm-Bragg theory is an ideal algorithm to handle cooperative processes. Here, we extend the analytical capabilities of the Zimm-Bragg theory in two directions. First, we combine the Zimm-Bragg partition function Z(T) with statistical-mechanical thermodynamics, explaining the thermodynamic system properties enthalpy, entropy and free energy with molecular parameters only. Second, the molecular enthalpy h0 to unfold a single amino acid residue is made temperaturedependent. The addition of a heat capacity term cv allows predicting not only heat denaturation, but also cold denaturation. Moreover, it predicts the heat capacity increase 0 p C ∆ in protein unfolding. The theory is successfully applied to differential scanning calorimetry experiments of proteins of different size and structure, that is, gpW62 (62aa), ubiquitin (74aa), lysozyme (129aa), metmyoglobin (153aa) and mAb monoclonal antibody (1290aa). Particular attention was given to the free energy, which can easily be obtained from the heat capacity Cp(T). The DSC experiments reveal a zero free energy for the native protein with an immediate decrease to negative free energies upon cold and heat denaturation. This trapezoidal shape is precisely reproduced by the Zimm-Bragg theory, whereas the so far applied non-cooperative 2-state model predicts a parabolic shape with a positive free energy maximum of the native protein. We demonstrate that the molecular parameters of the Zimm-Bragg theory have a well-defined physical meaning. In addition to predicting protein stability, independent of protein size, they yield estimates of unfolding kinetics and can be connected to molecular dynamics calculations.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2009

C 12 EO 8 (C 12 E 8) Triton X-100 Tween 80 We assessed the interaction of three electrically neut... more C 12 EO 8 (C 12 E 8) Triton X-100 Tween 80 We assessed the interaction of three electrically neutral detergents (Triton X-100, C 12 EO 8 , and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG tl 0 , that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergenttransporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG EO 0 = −1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.

Biochemistry, 1996

Three new analogues of the neuropeptide substance P (SP) were synthesized. The C-terminal message... more Three new analogues of the neuropeptide substance P (SP) were synthesized. The C-terminal message segment was made more hydrophilic in (Arg9)SP or more hydrophobic in (Nle9)SP. In (AcPro2, Arg9)SP the charge at the N-terminal address segment was reduced, while that of the message segment was increased. The rationale underlying these substitutions was to correlate the physical-chemical properties of the SP-analogues, in particular their lipid-induced conformation and membrane-binding affinity, with receptor binding and functional activity. In solution, all three analogues exhibited random coil conformations as evidenced by circular dichroism spectroscopy. Addition of SDS micelles induced partially alpha-helical structures. The same structure was also produced by negatively charged lipid vesicles for (AcPro2, Arg9)SP and (Arg9)SP whereas both alpha-helix-like structures and beta-sheet structures were observed for SP and (Nle9)SP. The measurement of the Gibbs adsorption isotherms and monolayer expansion studies provided quantitative data on the surface area requirement and on the membrane penetration area of the SP analogues. The thermodynamic parameters for lipid binding were determined with monolayer expansion for measurements and high-sensitivity titration calorimetry. The apparent binding constants, Kapp, for membranes containing 100% POPG were of the order of 10(3)- 10(5) M(-1). The binding was due to electrostatic attraction of the cationic peptides to the negatively charged membrane surface. The intrinsic (hydrophobic) binding constants, obtained after correcting for electrostatic effects, were much smaller with Kp=10+/- 1 M(-1) for (Arg9)SP, 9 +/- 1 M(-1) for (AcPro2, Arg9)SP, and 39 +/- 3 M(-1) for (Nle9)SP. The measurement of the binding affinities to the NK-1 receptor and of the in vitro activities showed that all three peptides behaved as agonists. Their binding affinity to the neurokinin-1 receptor decreased with the size of the side chains at position 9 of the amino acid sequence but was independent of the cationic charge of the peptides. The fact that even the highly charged (Arg9)SP has agonistic activity provides evidence that the binding epitope at the receptor is in a rather hydrophilic environment. This finding is in agreement with the low hydrophobic binding constants and the weak penetration of the three peptides into negatively charged membranes. It argues against a membrane mediated receptor mechanism and suggests that the agonist approaches the receptor binding, site from the aqueous phase.

ABC ATP-binding cassette (transport protein) MDR Multidrug resistance Pgp P-Glycoprotein (MDR1) (... more ABC ATP-binding cassette (transport protein) MDR Multidrug resistance Pgp P-Glycoprotein (MDR1) (Q)SAR (Quantitative) structure activity relationship TM Transmembrane (domain) H-bonding Hydrogen bonding Symbols A Strong H-bond acceptor (or strong electron donor) a Weak H-bond acceptor (or weak electron donor) ∆G aw Free Energy of partitioning into the air-water interface ∆G lw Free Energy of drug partitioning into the lipid membrane ∆G t Free Energy of drug binding to the transporter Pgp from within the lipid phase ∆G app Apparent Free Energy of binding to Pgp from the aqueous phase

Frontiers in Oncology

P-glycoprotein or multidrug resistance protein (MDR1) is an adenosine triphosphate (ATP) binding ... more P-glycoprotein or multidrug resistance protein (MDR1) is an adenosine triphosphate (ATP) binding cassette transporter (ABCB1) intensely investigated because it is an obstacle to successful pharmacotherapy of cancers. P-glycoprotein prevents cellular uptake of a large number of structurally and functionally diverse compounds, including most cancer therapeutics and in this way causes multidrug resistance (MDR). To overcome MDR, and thus improve cancer treatment, an understanding of P-glycoprotein inhibition at the molecular level is required. With this goal in mind, we propose rules that predict whether a compound is a modulator, substrate, inhibitor, or inducer of P-glycoprotein. This new set of rules is derived from a quantitative analysis of the drug binding and transport properties of P-glycoprotein. We further discuss the role of P-glycoprotein in immune surveillance and cell metabolism. Finally, the predictive power of the proposed rules is demonstrated with a set of FDA approved drugs which have been repurposed for cancer therapy.

ACS central science, Jan 22, 2017

ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a la... more ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a large variety of compounds across biological membranes. P-glycoprotein, involved in multidrug resistance, is the most investigated eukaryotic family member. Although a large number of biochemical and structural approaches have provided important information, the conformational dynamics underlying the coupling between ATP binding/hydrolysis and allocrite transport remains elusive. To tackle this issue, we performed molecular dynamic simulations for different nucleotide occupancy states of Sav1866, a prokaryotic P-glycoprotein homologue. The simulations reveal an outward-closed conformation of the transmembrane domain that is stabilized by the binding of two ATP molecules. The hydrolysis of a single ATP leads the X-loop, a key motif of the ATP binding cassette, to interfere with the transmembrane domain and favor its outward-open conformation. Our findings provide a structural basis for the ...

Molecular Pharmaceutics, 2016

The Journal of biological chemistry, Jan 12, 2016

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR, ABCC7), mutations of which ca... more The cystic fibrosis (CF) transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause CF, belongs to the ATP binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution to the total CFTR-related ATP hydrolysis rate (≥ 90 %) is due to phosphorylation by PKA and the minor contribution (≤ 10 %) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S which is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that...

Berichte Der Bunsengesellschaft Fur Physikalische Chemie, Nov 1, 1974

Phospholipiddoppelschichten sind die Grundbausteine vieler biologischer Membranen. Die Anordnung ... more Phospholipiddoppelschichten sind die Grundbausteine vieler biologischer Membranen. Die Anordnung und die Konformation der Lipid-ketten in der Doppelschicht konnte bisher nur mit Nitroxid-Spin-Labeln untersucht werden. Eine gleichwertige Methode ist Deuterium-Magnetische Resonanz von spezifisch deuterierten Phospholipiden. Die anisotrope Bewegung der Lipidketten fuhrt zu einer Aufspaltung des Deuterium-Signals in ein Dublett, wobei der Linienabstand in einfacher Weise mit dem Ordnungsgrad des deuterierten Kettensegments verknupft ist. Verglichen mit der Nitroxid-Spin-Label-Methode hat die Deuterium-Methode den Vorteil, das sie keine Storung in der Lipiddoppelschicht verursacht. Messungen mit spezifisch deuterierten L-α-Dipalmitoyllecithinen fuhren zu einem kooperativen Modell fur die Bewegung der Lipidketten. Phospholipid double layers are the basic structural units of many biological membranes. Till now, the order and conformation of the lipid chains in the double layer could only be studied by nitroxide spin labels. An equivalent method is deuterium magnetic resonance of specifically deuterated phospholipids. The anisotropic movement of the lipid chains gives rise to a doublet splitting of the deuterium signal. The distance of the lines is connected in a simple way with the degree of order of the deuterated chain segments. In comparison with the nitroxide spin label method, the deuterium method has the advantage of not disturbing the lipid double layer. Measurements with specifically deuterated L-α-dipalmitoyllecithins lead to a cooperative model of the movements of the lipid chains.

European Respiratory Journal, Sep 1, 2014

The rise of multi-drug resistant tuberculosis is a major threat. Multi drug efflux pumps are beli... more The rise of multi-drug resistant tuberculosis is a major threat. Multi drug efflux pumps are believed to significantly contribute to the development of resistance by reducing the intracellular concentration of the standard therapy, consisting of isoniazid (INH), ethambutol (ETH), rifampicin (RIF) and pyrazinamid (PZA). Immunohistochemical staining from a patient with tuberculosis of the lymphnodes revealed significant expression of P-gp and BCRP in macrophages at the site of infection. Next, we screened the interaction of tuberculostatics with P-glycoprotein (P-gp, ABCB1) and Breast Cancer Resistance Protein-1 (BCRP, ABCG2) over-expressed in plasma membranes using an ATPase activity assay. INH and PZA were found to be substrates for BCRP, substantially increasing its activity at clinical concentrations. Moreover, the antimalarial chloroquine inhibited both pumps at clinically relevant concentrations. Differentiated THP-1macropaghes were shown to significantly increase the expression of the respective pumps upon stimulation with interferon (IFN)-γ. In a functional assay, the efflux of INH was reduced in the presence of therapeutic concentrations of chloroquine, as measured in the supernatant with liquid chromatography–high resolution mass spectrometry. This effect was clearly more pronounced in IFN-γ stimulated macrophages. In conclusion, we showed for the first time that INH and PZA are substrates for BCRP. IFN-γ stimulated cells pumped INH out into the extracellular space, an effect which could be reversed by chloroquine. Altogether chloroquine, a safe and well-tolerated drug with a long half-life could enhance intracellular tuberculostatic activity, potentially shortening treatment duration.

Bioorganic & Medicinal Chemistry Letters, 2006

7-Deoxypaclitaxel, 10-deacetoxypaclitaxel and 10-deacetoxy-7-deoxypaclitaxel were prepared and ev... more 7-Deoxypaclitaxel, 10-deacetoxypaclitaxel and 10-deacetoxy-7-deoxypaclitaxel were prepared and evaluated for their ability to promote assembly of tubulin into microtubules, their cytotoxicity against NCI/ADR-RES cells and for their interactions with Pglycoprotein in bovine brain microvessel endothelial cells. The three compounds were essentially equivalent to paclitaxel in cytotoxicity against NCI/ADR-RES cells. They also appeared to interact with P-glycoprotein in the endothelial cells with the two 10-deacetoxy compounds having less interaction than paclitaxel and 7-deoxypaclitaxel.

Protein unfolding is a dynamic cooperative process with many short-lived intermediates. Cooperati... more Protein unfolding is a dynamic cooperative process with many short-lived intermediates. Cooperativity means that similar molecular elements act dependently on each other. The thermodynamics of protein unfolding can be determined with differential scanning calorimetry (DSC). The measurement of the heat capacity provides the temperature profiles of enthalpy, entropy and free energy. The thermodynamics of protein unfolding is completely determined with these thermodynamic properties. We emphasise the model-independent analysis of the heat capacity. The temperature profiles of enthalpy H(T), entropy S(T) and free energy G(T) can be obtained directly by a numerical integration of Cp(T). In evaluating different models for protein unfolding. It is essential to simulate all thermodynamic properties, not only the heat capacity. A chemical equilibrium two-state model is a widely used approximation to protein unfolding. The model assumes a chemical equilibrium between only two protein conforma...

Methods and Principles in Medicinal Chemistry, 2004

Seminars in Cancer Biology, 1997

We review how P-glycoprotein recognizes a wide variety of compounds and how it carries its substr... more We review how P-glycoprotein recognizes a wide variety of compounds and how it carries its substrates across membranes. Amino acid substitutions that affect the substrate specificity of P-glycoprotein have been found scattered throughout the molecule. In particular, some amino acid () residues in the putative transmembrane domain TM 1 together with TM5᎐6 and TM11᎐12 may help to govern substrate specificity. The features that substrates for P-glycoprotein share are also discussed. The amphipathy of a substrate may decide whether the substrate can be intercalated into the lipid bilayer of the membrane. In addition, only certain molecular volumes and tertiary structures may make it possible for the substrate to fit into the substrate-() binding site s of P-glycoprotein.

Bioorganic & Medicinal Chemistry Letters, 2006

A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core tha... more A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core that reduces interaction with P-glycoprotein in bovine brain microvessel endothelial cells is described. Modification and derivatization of the C10 po\sition were carried out using a substrate controlled hydride addition to a key C9 and C10 diketone intermediate. The analogs were tested for tubulin assembly and cytotoxicity, and were shown to retain potency similar to paclitaxel. P-glycoprotein interaction was examined using a rhodamine assay and it was found that simple hydrolysis or epimerization of the C10 acetate of paclitaxel and Taxol C can reduce interaction with the P-glycoprotein transporter that may allow for increased permeation of taxanes into the brain.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990

The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gin-Phe-Phe-Gly-Leu-Met amide) ... more The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gin-Phe-Phe-Gly-Leu-Met amide) and three of its antagonists were derived by measuring the Gibbs adsorption isotherm, providing information on the surface activity, the molecular shape, and the pK values of the different molecules. The following three antagonists were investigated: [

Protein stability is of utmost importance in many areas of life sciences and life science technol... more Protein stability is of utmost importance in many areas of life sciences and life science technology. The gold standard for assessing protein stability is the measurement of the heat capacity Cp(T), showing large peaks at the temperature of cold and heat unfolding. We first demonstrate that important thermodynamic properties, including the free energy, can be obtained from Cp(T) temperature profiles without the necessity of an unfolding model. This is illustrated with experimental examples taken from the literature. We then compare all thermodynamic results with chemical and statistical unfolding models. An often-cited chemical equilibrium two-state model makes partially incorrect predictions. New models are introduced, which are in excellent agreement with the experimental results. We then show that the free energy is not a good criterion to judge protein stability. More useful parameters are discussed, including protein cooperativity. The new parameters are embedded in a well-defi...

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987

The bindln~ of the local anesthetic dilmcaine to myers composed of 1-paimitoyl-2-uleoyl-sn-giyeer... more The bindln~ of the local anesthetic dilmcaine to myers composed of 1-paimitoyl-2-uleoyl-sn-giyeero-3plmsplmclmline was studied with a Langmuir trough at pH 5.5 (22°C, 0.1 M NaO). At this pH value only the charged form of the local anesthetic exists in solution. Charged dibucaine was found to be surface active and to penetrate into the lipid monolayer, with the hydrophobic part of the molecule being accommodated between the fatty acyl chains of the Hpid. The dibucaine intercalation could be quantitated by meastwing the expansion of the film area, AA, at constant surface pressme, yr. At a given surface pressure, AA increased with increasing dilmcaine in the buffer phase. On the other hand, keeping the dibucaine concentration constant, the area increase, AA, was strongly dependent on the surface pressure. The area increase, AA, was large at low surface pressure and decreased with increasing sm~ace pressure. A plot of the relative change in surface area, /tA/A, versus the surface pressure yielded straight lines in the pressure range of 25-36 mN/m for five different concentrations. The AA/A vs. vr isotherms intersected at vr-39.5 + 1 mN/m with AA =0, indicating that charged dilmcaine apparentiy can no longer penetrate into the monolayer film. By making judicial assumptions about the area requirement of dilmcaine the monolayer expansion curves could be transformed into true binding isotherms. Dibucaine binding isotherms were constructed for different monolayer pressures and were compared to a bilayer binding isotherm measured under similar conditions with ultraviolet spectroscopy. The best agreement between monolayer and bilayer binding data was obtained for a monolayer held at a pressure of 30.7 to 32.5 raN/m, which can thus be considered as the bilayer-monolayer equivalence lW&,sure. It is further suggested from this analogy that the binding of dibueaine does not change the internal pressure in the bilayer phase, at least not in the concentration range of physiological interest (0-2 mM dilmcaiue) but induces a lateral expansion. At higher molar ratios of cationic dilmeaine to lipid, Xb, in the monolayer (x b >0.20) the area increase is larger than would be expected from the molecular dimensions of dibueaine. This is probably due to charge repulsion effects, which at still higher molar ratios (x b > 0.6) lead to a micemsation. The pressure dependence of the intercalation of cationic dibueaine into lipid membranes may also be of relevance for the phenomenon of pressure reversal in anesthesia.

SSRN Electronic Journal, 2021

Protein unfolding is a dynamic cooperative equilibrium between short lived protein conformations.... more Protein unfolding is a dynamic cooperative equilibrium between short lived protein conformations. The Zimm-Bragg theory is an ideal algorithm to handle cooperative processes. Here, we extend the analytical capabilities of the Zimm-Bragg theory in two directions. First, we combine the Zimm-Bragg partition function Z(T) with statistical-mechanical thermodynamics, explaining the thermodynamic system properties enthalpy, entropy and free energy with molecular parameters only. Second, the molecular enthalpy h0 to unfold a single amino acid residue is made temperaturedependent. The addition of a heat capacity term cv allows predicting not only heat denaturation, but also cold denaturation. Moreover, it predicts the heat capacity increase 0 p C ∆ in protein unfolding. The theory is successfully applied to differential scanning calorimetry experiments of proteins of different size and structure, that is, gpW62 (62aa), ubiquitin (74aa), lysozyme (129aa), metmyoglobin (153aa) and mAb monoclonal antibody (1290aa). Particular attention was given to the free energy, which can easily be obtained from the heat capacity Cp(T). The DSC experiments reveal a zero free energy for the native protein with an immediate decrease to negative free energies upon cold and heat denaturation. This trapezoidal shape is precisely reproduced by the Zimm-Bragg theory, whereas the so far applied non-cooperative 2-state model predicts a parabolic shape with a positive free energy maximum of the native protein. We demonstrate that the molecular parameters of the Zimm-Bragg theory have a well-defined physical meaning. In addition to predicting protein stability, independent of protein size, they yield estimates of unfolding kinetics and can be connected to molecular dynamics calculations.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2009

C 12 EO 8 (C 12 E 8) Triton X-100 Tween 80 We assessed the interaction of three electrically neut... more C 12 EO 8 (C 12 E 8) Triton X-100 Tween 80 We assessed the interaction of three electrically neutral detergents (Triton X-100, C 12 EO 8 , and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG tl 0 , that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergenttransporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG EO 0 = −1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.

Biochemistry, 1996

Three new analogues of the neuropeptide substance P (SP) were synthesized. The C-terminal message... more Three new analogues of the neuropeptide substance P (SP) were synthesized. The C-terminal message segment was made more hydrophilic in (Arg9)SP or more hydrophobic in (Nle9)SP. In (AcPro2, Arg9)SP the charge at the N-terminal address segment was reduced, while that of the message segment was increased. The rationale underlying these substitutions was to correlate the physical-chemical properties of the SP-analogues, in particular their lipid-induced conformation and membrane-binding affinity, with receptor binding and functional activity. In solution, all three analogues exhibited random coil conformations as evidenced by circular dichroism spectroscopy. Addition of SDS micelles induced partially alpha-helical structures. The same structure was also produced by negatively charged lipid vesicles for (AcPro2, Arg9)SP and (Arg9)SP whereas both alpha-helix-like structures and beta-sheet structures were observed for SP and (Nle9)SP. The measurement of the Gibbs adsorption isotherms and monolayer expansion studies provided quantitative data on the surface area requirement and on the membrane penetration area of the SP analogues. The thermodynamic parameters for lipid binding were determined with monolayer expansion for measurements and high-sensitivity titration calorimetry. The apparent binding constants, Kapp, for membranes containing 100% POPG were of the order of 10(3)- 10(5) M(-1). The binding was due to electrostatic attraction of the cationic peptides to the negatively charged membrane surface. The intrinsic (hydrophobic) binding constants, obtained after correcting for electrostatic effects, were much smaller with Kp=10+/- 1 M(-1) for (Arg9)SP, 9 +/- 1 M(-1) for (AcPro2, Arg9)SP, and 39 +/- 3 M(-1) for (Nle9)SP. The measurement of the binding affinities to the NK-1 receptor and of the in vitro activities showed that all three peptides behaved as agonists. Their binding affinity to the neurokinin-1 receptor decreased with the size of the side chains at position 9 of the amino acid sequence but was independent of the cationic charge of the peptides. The fact that even the highly charged (Arg9)SP has agonistic activity provides evidence that the binding epitope at the receptor is in a rather hydrophilic environment. This finding is in agreement with the low hydrophobic binding constants and the weak penetration of the three peptides into negatively charged membranes. It argues against a membrane mediated receptor mechanism and suggests that the agonist approaches the receptor binding, site from the aqueous phase.

ABC ATP-binding cassette (transport protein) MDR Multidrug resistance Pgp P-Glycoprotein (MDR1) (... more ABC ATP-binding cassette (transport protein) MDR Multidrug resistance Pgp P-Glycoprotein (MDR1) (Q)SAR (Quantitative) structure activity relationship TM Transmembrane (domain) H-bonding Hydrogen bonding Symbols A Strong H-bond acceptor (or strong electron donor) a Weak H-bond acceptor (or weak electron donor) ∆G aw Free Energy of partitioning into the air-water interface ∆G lw Free Energy of drug partitioning into the lipid membrane ∆G t Free Energy of drug binding to the transporter Pgp from within the lipid phase ∆G app Apparent Free Energy of binding to Pgp from the aqueous phase

Frontiers in Oncology

P-glycoprotein or multidrug resistance protein (MDR1) is an adenosine triphosphate (ATP) binding ... more P-glycoprotein or multidrug resistance protein (MDR1) is an adenosine triphosphate (ATP) binding cassette transporter (ABCB1) intensely investigated because it is an obstacle to successful pharmacotherapy of cancers. P-glycoprotein prevents cellular uptake of a large number of structurally and functionally diverse compounds, including most cancer therapeutics and in this way causes multidrug resistance (MDR). To overcome MDR, and thus improve cancer treatment, an understanding of P-glycoprotein inhibition at the molecular level is required. With this goal in mind, we propose rules that predict whether a compound is a modulator, substrate, inhibitor, or inducer of P-glycoprotein. This new set of rules is derived from a quantitative analysis of the drug binding and transport properties of P-glycoprotein. We further discuss the role of P-glycoprotein in immune surveillance and cell metabolism. Finally, the predictive power of the proposed rules is demonstrated with a set of FDA approved drugs which have been repurposed for cancer therapy.

ACS central science, Jan 22, 2017

ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a la... more ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a large variety of compounds across biological membranes. P-glycoprotein, involved in multidrug resistance, is the most investigated eukaryotic family member. Although a large number of biochemical and structural approaches have provided important information, the conformational dynamics underlying the coupling between ATP binding/hydrolysis and allocrite transport remains elusive. To tackle this issue, we performed molecular dynamic simulations for different nucleotide occupancy states of Sav1866, a prokaryotic P-glycoprotein homologue. The simulations reveal an outward-closed conformation of the transmembrane domain that is stabilized by the binding of two ATP molecules. The hydrolysis of a single ATP leads the X-loop, a key motif of the ATP binding cassette, to interfere with the transmembrane domain and favor its outward-open conformation. Our findings provide a structural basis for the ...

Molecular Pharmaceutics, 2016

The Journal of biological chemistry, Jan 12, 2016

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR, ABCC7), mutations of which ca... more The cystic fibrosis (CF) transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause CF, belongs to the ATP binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution to the total CFTR-related ATP hydrolysis rate (≥ 90 %) is due to phosphorylation by PKA and the minor contribution (≤ 10 %) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S which is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that...

Berichte Der Bunsengesellschaft Fur Physikalische Chemie, Nov 1, 1974

Phospholipiddoppelschichten sind die Grundbausteine vieler biologischer Membranen. Die Anordnung ... more Phospholipiddoppelschichten sind die Grundbausteine vieler biologischer Membranen. Die Anordnung und die Konformation der Lipid-ketten in der Doppelschicht konnte bisher nur mit Nitroxid-Spin-Labeln untersucht werden. Eine gleichwertige Methode ist Deuterium-Magnetische Resonanz von spezifisch deuterierten Phospholipiden. Die anisotrope Bewegung der Lipidketten fuhrt zu einer Aufspaltung des Deuterium-Signals in ein Dublett, wobei der Linienabstand in einfacher Weise mit dem Ordnungsgrad des deuterierten Kettensegments verknupft ist. Verglichen mit der Nitroxid-Spin-Label-Methode hat die Deuterium-Methode den Vorteil, das sie keine Storung in der Lipiddoppelschicht verursacht. Messungen mit spezifisch deuterierten L-α-Dipalmitoyllecithinen fuhren zu einem kooperativen Modell fur die Bewegung der Lipidketten. Phospholipid double layers are the basic structural units of many biological membranes. Till now, the order and conformation of the lipid chains in the double layer could only be studied by nitroxide spin labels. An equivalent method is deuterium magnetic resonance of specifically deuterated phospholipids. The anisotropic movement of the lipid chains gives rise to a doublet splitting of the deuterium signal. The distance of the lines is connected in a simple way with the degree of order of the deuterated chain segments. In comparison with the nitroxide spin label method, the deuterium method has the advantage of not disturbing the lipid double layer. Measurements with specifically deuterated L-α-dipalmitoyllecithins lead to a cooperative model of the movements of the lipid chains.

European Respiratory Journal, Sep 1, 2014

The rise of multi-drug resistant tuberculosis is a major threat. Multi drug efflux pumps are beli... more The rise of multi-drug resistant tuberculosis is a major threat. Multi drug efflux pumps are believed to significantly contribute to the development of resistance by reducing the intracellular concentration of the standard therapy, consisting of isoniazid (INH), ethambutol (ETH), rifampicin (RIF) and pyrazinamid (PZA). Immunohistochemical staining from a patient with tuberculosis of the lymphnodes revealed significant expression of P-gp and BCRP in macrophages at the site of infection. Next, we screened the interaction of tuberculostatics with P-glycoprotein (P-gp, ABCB1) and Breast Cancer Resistance Protein-1 (BCRP, ABCG2) over-expressed in plasma membranes using an ATPase activity assay. INH and PZA were found to be substrates for BCRP, substantially increasing its activity at clinical concentrations. Moreover, the antimalarial chloroquine inhibited both pumps at clinically relevant concentrations. Differentiated THP-1macropaghes were shown to significantly increase the expression of the respective pumps upon stimulation with interferon (IFN)-γ. In a functional assay, the efflux of INH was reduced in the presence of therapeutic concentrations of chloroquine, as measured in the supernatant with liquid chromatography–high resolution mass spectrometry. This effect was clearly more pronounced in IFN-γ stimulated macrophages. In conclusion, we showed for the first time that INH and PZA are substrates for BCRP. IFN-γ stimulated cells pumped INH out into the extracellular space, an effect which could be reversed by chloroquine. Altogether chloroquine, a safe and well-tolerated drug with a long half-life could enhance intracellular tuberculostatic activity, potentially shortening treatment duration.