Jose A. Ferragut

Altered Drug Membrane Permeability in a Multidrug- Resistant Leishmania tropica Line

We selected a Leishmania tropica cell line resistant to daunomycin (DNM) that presents a multidrug-resistant (MDR) phenotype characterized by overexpression of a P-glycoprotein of 150 kDa. The resistant line overexpressed an MDR-like gene, called ltrmdr1, located in an extrachromosomal circular DNA. DNM uptake experiments using laser flow cytometry showed a significant reduction in drug accumulation in the resistant parasites. The initial stages of the interaction of DNM with membranes from wild-type and DNM-resistant parasites were defined by a rapid kinetic stopped-flow procedure which can be described by two kinetic components. On the basis of a previous similar kinetic study with tumor cells, we ascribed the fast component to rapid interaction of DNM with membrane surface components and the slow component to passive diffusion of the drug across the membranes. The results reported here indicate that entrance of DNM into wild-type parasites was facilitated in respect to the resistant ones. We propose that resistance to DNM in L. tropica is a multifactorial event involving at least two complementary mechanisms. an altered drug membrane permeability and the overexpression of a protein related to P-glycoprotein that regulates drug efflux.

Susceptibility of multidrug resistance tumor cells to apoptosis induction by histone deacetylase inhibitors.

The main goal of our study has been to analyze the efficiency of new anticancer drugs, specifically histone deacetylase inhibitors, in tumor cells bearing a multidrug resistance phenotype. We report that the histone deacetylase inhibitors, Trichostatin A and Suberoylanilide Hydroxamic Acid (SAHA), dramatically reduce cell viability and promote apoptosis in different drug‐resistant cells, affecting in a much lesser extent to their parental drug‐sensitive counterparts. The differential effects induced by Trichostatin A and SAHA between drug‐sensitive and drug‐resistant cells are reflected on the main characteristics of the resistant phenotype. Thus, reverse transcription‐PCR and Western immunoblots confirm that both histone deacetylase inhibitors promote endogenous down‐regulation of P‐glycoprotein, which is overexpressed in the drug‐resistant cells. Transfection of drug‐sensitive cells with the P‐glycoprotein cDNA ruled out the a priori possible association between apoptosis and down‐regulation of P‐glycoprotein induced by the histone deacetylase inhibitors. The results suggest a therapeutic potential of histone deacetylase inhibitors in the treatment of cancers with acquired resistance.

Labeling of the nicotinic acetylcholine receptor by a photoactivatable steroid probe : effects of cholesterol and cholinergic ligands

A photoactivatable steroid, p-azidophenacyl 3 alpha-hydroxy-5 beta-cholan-24- ate (APL), has been synthesized and used instead of cholesterol to functionally reconstitute purified acetylcholine receptor (AcChR) into vesicles made of asolectin phospholipids. Upon irradiation, the extent of AcChR photolabeling by APL is directly proportional to the amount of APL incorporated into the reconstituted vesicles and the maximum stoichiometry observed corresponds to approx. 50 mol of APL bound per mol of AcChR. Furthermore, all four subunits of the AcChR become labeled by APL and the observed labeling pattern resembles the 2:1:1:1 stoichiometry characteristic of these subunits within the AcChR complex. The presence of either cholesterol or neutral lipids from asolectin in the reconstituted bilayer decreases both, the incorporation of APl into the vesicles and the covalent labeling of the AcChR upon irradiation, without altering the stoichiometry of labeling in AcChR subunits stated above. This suggests that the potential interaction sites for the photoactivatable probe in the reconstituted AcChR are mostly those normally occupied by the natural neutral lipids. Carbamylcholine, a cholinergic agonist, also reduces the extent of APL photolabeling of the AcChR in a dose-dependent manner but, in contrast to the effects of cholesterol, the presence of carbamylcholine alters the stoichiometry of labeling in the AcChR subunits. This, along with the observation that such a decrease in the extent of APL photolabeling caused by carbamylcholine can be blocked by preincubation with alpha-bungarotoxin, suggest that AcChR desensitization induced by prolonged exposure to cholinergic agonists encompasses a rearrangement of transmembrane portions of the AcChR protein, which can be sensed by the photoactivatable probe. Conversely, presence of (+)-tubocurarine, a competitive cholinergic antagonist, has no effects on altering either the extent of APL photolabeling of the AcChR or the distribution of the labeling among AcChR subunits.

Phospholipase A2 hydrolysis of membrane phospholipids causes structural alteration of the nicotinic acetylcholine receptor

Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the alpha-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of alpha-bungarotoxin-binding sites are shifted to lower temperatures by approx. 7 and 5 C degrees, respectively, upon exposure to phospholipase A2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3(n-3] causes detectable destabilization of the alpha-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A2 hydrolysis products.

Experimental Resistance to Drug Combinations in Leishmania donovani: Metabolic and Phenotypic Adaptations

ABSTRACT Together with vector control, chemotherapy is an essential tool for the control of visceral leishmaniasis (VL), but its efficacy is jeopardized by growing resistance and treatment failure against first-line drugs. To delay the emergence of resistance, the use of drug combinations of existing antileishmanial agents has been tested systematically in clinical trials for the treatment of visceral leishmaniasis (VL). In vitro, Leishmania donovani promastigotes are able to develop experimental resistance to several combinations of different antileishmanial drugs after 10 weeks of drug pressure. Using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach, we identified metabolic changes in lines that were experimentally resistant to drug combinations and their respective single-resistant lines. This highlighted both collective metabolic changes (found in all combination therapy-resistant [CTR] lines) and specific ones (found in certain CTR lines). We demonstrated that single-resistant and CTR parasite cell lines show distinct metabolic adaptations, which all converge on the same defensive mechanisms that were experimentally validated: protection against drug-induced and external oxidative stress and changes in membrane fluidity. The membrane fluidity changes were accompanied by changes in drug uptake only in the lines that were resistant against drug combinations with antimonials, and surprisingly, drug accumulation was higher in these lines. Together, these results highlight the importance and the central role of protection against oxidative stress in the different resistant lines. Ultimately, these phenotypic changes might interfere with the mode of action of all drugs that are currently used for the treatment of VL and should be taken into account in drug development.

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier‐transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide 1 infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist‐induced alterations by band‐fitting analysis of the amide 1 spectral band reveals no appreciable changes in the percent of α‐helix, but a decrease in β‐sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration‐dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinerpic agonist implies significant rearrangements in the protein structure.

Structural stabilization of botulinum neurotoxins by tyrosine phosphorylation.

Tyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal stability, suggesting a substantial modification of the global protein conformation. We used Fourier‐transform infrared (FTIR) spectroscopy to study changes of secondary structure and thermostability of tyrosine phosphorylated botulinum neurotoxins A (BoNT A) and E (BoNT E). Changes in the conformationally‐sensitive amide I band upon phosphorylation indicated an increase of the α‐helical content with a concomitant decrease of less ordered structures such as turns and random coils, and without changes in β‐sheet content. These changes in secondary structure were accompanied by an increase in the residual amide II absorbance band remaining upon H‐D exchange, consistent with a tighter packing of the phosphorylated proteins. FTIR and differential scanning calorimetry (DSC) analyses of the denaturation process show that phosphorylated neurotoxins denature at temperatures higher than those required by non‐phosphorylated species. These findings indicate that tyrosine phosphorylation induced a transition to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and thermostability.

Cholesterol stabilizes the structure of the nicotinic acetylcholine receptor reconstituted in lipid vesicles

Abstract A technique of heat inactivation of α-bungarotoxin binding sites, has been used to probe structural alteration of the nicotinic acetylcholine receptor when reconstituted into soybean lipid vesicles containing different amounts of added cholesterol. The profiles of heat inactivation of α-bungarotoxin binding sites are gradually shifted to higher temperatures, as the cholesterol/phospholipid molar ratio in the reconstituted vesicles is increased from 0 to 0.4, thus, indicating that presence of cholesterol within the lipid matrix produces a structural stabilization of the reconstituted acetylcholine receptor protein. The observed stabilization of receptor structure induced by cholesterol is such that, depending upon the different conditions used to form the reconstituted vesicles by detergent dialysis procedures, the profiles of heat inactivation for the reconsutituted receptor vesicles at a cholesterol/phospholipid molar ratio of 0.4 become undistinguishable from that exhibited by native acetylcholine receptor membranes isolated from the electric organ of Torpedo . Increasing the cholesterol concentration in the reconstituted vesicles also induces a decrease in the apparent ‘fluidity’ of the membrane, which correlates very closely with the observed stabilization of the receptor protein. Such a correlation, however, does not necessarily imply that changes in receptor structure are caused by pertubations of the membrane ‘fluidity’. This conclusion is based on experiments using local anesthetics, well known to cause alteration of membrane lipid dynamics, but unable to modify the characteristic heat-inactivation profiles from native acetylcholine receptor membranes. As a possible alternative to the above observations, it is suggested that the effects of cholesterol on receptor structure could be exerted through direct interaction with the receptor protein. Also, since similarly high concentrations of cholesterol have been reported to be required for optimal cation-gating activity of reconstituted acetylcholine receptor, we interpret our data as indicative of a correlation between structural and functional alterations of the acetylcholine receptor induced by the presence of cholesterol within the membrane bilayer.

Ultrastructural alterations in plasma membranes from drug-resistant P388 murine leukemia cells

Freeze-fracture studies of daunomycin-sensitive and daunomycin-resistant P388 cell lines, reveal a significant increase in the numerical density of intramembrane particles at both, the protoplasmic and the exoplasmic leaflets of the plasma membrane from the drug-resistant cells. Such change in plasma membrane architecture is not accompanied by overexpression of P-glycoproteins. Furthermore, drug-sensitive cells exhibited an increased number of exo-endocytotic images when compared to drug-resistant cells. Our observations suggest that there are global changes in the structural organization of the plasma membrane, which are related to the acquisition of the cellular drug-resistant phenotype.

Rapid kinetics of the interaction between daunomycin and drug-sensitive or drug-resistant P388 leukemia cells

The initial stages of the interaction of daunomycin (DNM) with drug‐sensitive (P388/S) and drug‐resistant (P388/100) cells have been defined by a rapid kinetics stopped‐flow procedure. The process can be described by two kinetic components. The faster component accounts for rapid occupation of cell surface sites by DNM, as supported by experiments with liposomes with different surface charge. On the other hand, the effect of verapamil in the assays, suggests that the slower component is involved in the transport of the drug into the cells. Our observations are consistent with a loss in the control of the passive permeability to the drugs in the drug‐resistant tumor cells.