High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease.

African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.

A new ABC half-transporter in Leishmania major is involved in resistance to antimony.

The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes.

Dihydro-beta-agarofuran sesquiterpenes: a new class of reversal agents of the multidrug resistance phenotype mediated by P-glycoprotein in the protozoan parasite Leishmania.

Leishmaniasis is the most important emerging and uncontrolled infectious disease and the second cause of death among parasitic diseases, after Malaria. One of the main problems concerning the control of infectious diseases is the increased resistance to usual drugs. Overexpression of P-glycoprotein (Pgp)-like transporters represents a very efficient mechanism to reduce the intracellular accumulation of drugs in cancer cells and parasitic protozoans, thus conferring a multidrug resistance (MDR) phenotype. Pgps are active pumps belonging to the ATP-binding cassette (ABC) superfamily of proteins. The inhibition of the activity of these proteins represents an interesting way to control drug resistance both in cancer and in infectious diseases. Most conventional mammalian Pgp-MDR modulators are ineffective in the modulation of Pgp activity in the protozoan parasite Leishmania. Consequently, there is a necessity to find effective modulators of Pgp-MDR for protozoan parasites. In this review we describe a rational strategy developed to find specific Pgp-MDR modulators in Leishmania, using natural and semisynthetic dihydro-beta-agarofuran sesquiterpenes from Celastraceae plants. A series of these compounds have been tested on a MDR Leishmania tropica line overexpressing a Pgp transporter to determine their ability to revert the resistance phenotype and to modulate intracellular drug accumulation. Almost all of these natural compounds showed potent reversal activity with different degrees of selectivity and a significant low toxicity. The three-dimensional quantitative structure-activity relationship using the comparative molecular similarity indices analysis (CoMSIA), was employed to characterize the requirements of these sesquiterpenes as modulators at Pgp-like transporter in Leishmania.

RU49953: a non-hormonal steroid derivative that potently inhibits P-glycoprotein and reverts cellular multidrug resistance.

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

Multidrug resistance phenotype mediated by the P-glycoprotein-like transporter in Leishmania: a search for reversal agents.

Abstract: Protozoan parasites are responsible for important diseases that threaten the lives of nearly one-quarter of the human population world-wide. Among them, leishmaniasis has become the second cause of death, mainly due to the emergence of parasite resistance to conventional drugs. P-glycoprotein (Pgp)-like transporters overexpression is a very efficient mechanism to reduce the intracellular accumulation of many drugs in cancer cells and parasitic protozoans including Plasmodium and Leishmania, thus conferring a multidrug resistance (MDR) phenotype. Therefore, there is a great clinical interest in developing inhibitors of these transporters to overcome such a resistance. Pgps are active pumps belonging to the ATP-binding cassette (ABC) superfamily of proteins, and consist of two homologous halves, each containing a transmembrane domain (TMD) involved in drug efflux, and a cytosolic nucleotide-binding domain (NBD) responsible for ATP binding and hydrolysis. Most conventional cancer MDR modulators interact with the drug-binding sites on the TMDs of Pgps, but they are also usually transported and the required concentrations for a permanent inhibition produce subsequent side-effects that hamper their clinical use. Besides, they only poorly modulate the resistance in protozoan parasites. We review here a rational strategy developed to overcome the MDR phenotype in Leishmania, consisting in: i) the selection of an MDR Leishmania tropica line that overexpresses a Pgp-like transporter; ii) the use of their cytosolic NBDs as new pharmacological targets; iii) the search of new natural compounds that revert the MDR phenotype in Leishmania by binding to the TMDs; iv) the combination of subdoses of the above selected modulators directed to both targets in the transporter, NBDs and TMDs, to accumulate their reversal effects while diminishing their toxicity. In this way, we have reverted the MDR phenotype in Leishmania, including the resistance to the most promising new antileishmania agents, the alkyl-lysophospholipids. This approach might be extrapolated to be used in other eukaryotic cells.

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

Chemosensitization of a multidrug-resistant Leishmania tropica line by new sesquiterpenes from Maytenus magellanica and Maytenus chubutensis.

Parasite resistance to drugs has emerged as a major problem in current medicine, and therefore, there is great clinical interest in developing compounds that overcome these resistances. In an intensive study of South American medicinal plants, herein we report the isolation, structure elucidation, and biological activity of dihydro-beta-agarofuran sesquiterpenes from the roots of Maytenus magellanica (1-14) and M. chubutensis (14-17). This type of natural products may be considered as privileged structures. The structures of 10 new compounds, 1, 3, 6-9, and12-15, were determined by means of (1)H and (13)C NMR spectroscopic studies, including homonuclear (COSY and ROESY) and heteronuclear correlation experiments (HMQC and HMBC). The absolute configurations of eight hetero- and homochromophoric compounds, 1, 3,6-9, 12, and 13, were determined by means of CD studies. Fourteen compounds, 1-3 and 6-16, have been tested on a multidrug-resistant Leishmania tropica line overexpressing a P-glycoprotein-like transporter to determine their ability to revert the resistance phenotype and to modulate intracellular drug accumulation. From this series, 1, 2, 3, 14, and 15 showed potent activity, 1 being the most active compound. The structure-activity relationships of the different compounds are discussed.

Alkyl-lysophospholipid resistance in multidrug-resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like transporter modulator.

Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C(5)-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.

High-affinity binding of silybin derivatives to the nucleotide-binding domain of a Leishmania tropica P-glycoprotein-like transporter and chemosensitization of a multidrug-resistant parasite to daunomycin.

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters.

ABC transporters in the protozoan parasite Leishmania.

ATP-binding cassette (ABC) transporters constitute one of the biggest and most conserved protein families in the evolutionary scale. Many of them are of enormous clinical relevance, due to their relationship with genetic diseases and drug resistance during the treatment of cancer and infectious diseases. Leishmaniasis is a major and globally widespread group of parasitic diseases, whose treatment has been complicated by the expansion of resistance to conventional drugs. Here, we review the current knowledge about ABC transporters in Leishmania spp, with special attention to their relationship with the drug-resistance phenotype.

Evolutionary relationships in Trypanosoma cruzi: molecular phylogenetics supports the existence of a new major lineage of strains.

For the purpose of investigating the evolutionary relationships among strains of the human parasite Trypanosoma cruzi, we have determined the nucleotide sequence, in 16 T. cruzi stocks, of a DNA fragment having approximately 1030 nucleotides in length. Phylogenetic analyses show the presence of at least three major groups of T. cruzi strains, a result that contradicts previous phylogenetic inferences based on polymorphism data. We also performed an analysis of the relative extent of nucleotide divergence among T. cruzi strains compared to the divergence between Leishmania species, using the gene encoding pteridine reductase. The results presented in this work show that the divergence among the most distant T. cruzi strains is at least as high as the divergence between two different species complexes of Leishmania, those containing L. major and L. mexicana.

Trypanosoma cruzi: molecular cloning of a gene coding for a putative vacuolar protein.

We describe the characterization of Tc38, a Trypanosoma cruzi gene coding for a 337-amino-acid protein with a predicted molecular mass of 38 kDa. Tc38 presents similarities to the plant storage vacuolar protein gamma-3-hordein involved in the transport and targeting of prolamins to the vacuole of developing barley endosperm. Western blot analysis using a polyclonal antiserum against recombinant Tc38 revealed that the protein is differentially expressed in the different life stages of the parasite, showing a higher expression in the epimastigote and tripomastigote stages. Immunofluorescence studies suggest that the protein is located in putative vacuolar structures in epimastigotes. The functionality of this protein in T. cruzi remains to be elucidated.

New natural sesquiterpenes as modulators of daunomycin resistance in a multidrug-resistant Leishmania tropica line.

The effects produced by nine dihydro-beta-agarofuran sesquiterpenes isolated from Crossopetalum tonduzii (1-8) and Maytenus macrocarpa (9) (Celastraceae) on the reversion of the resistant phenotype on a multidrug-resistant Leishmania line and their binding to recombinant C-terminal nucleotide-binding domain of Leishmania P-glycoprotein-like transporter were studied. The structures of the new compounds (1-5) were elucidated by spectroscopic methods, including (1)H-(13)C heteronuclear correlation (HMQC), long-range correlation spectra with inversal detection (HMBC), ROESY experiments, and chemical correlations. The absolute configuration of one of them (1) was determined by CD studies. The structure-activity relationship is discussed.

Correlation between the affinity of flavonoids binding to the cytosolic site of Leishmania tropica multidrug transporter and their efficiency to revert parasite resistance to daunomycin.

The C-terminal nucleotide-binding domain (NBD2) of a P-glycoprotein-like transporter, encoded by the ltrmdr1 gene in Leishmania tropica and involved in parasite multidrug resistance (MDR), was overexpressed in Escherichia coli as a hexahistidine tagged protein and purified. The L. tropica recombinant domain efficiently bound fluorescent derivatives of ATP, the hydrophobic steroid analogue RU 486, and different classes of flavonoids with the following efficiency: flavone > flavanone > isoflavone > glucorhamnosyl-flavone > chromone. The affinity for flavones was dependent on the presence of hydroxyl groups at positions 5 and 3 and was further increased by a hydrophobic 1,1-dimethylallyl substituent at position 8. When flow cytometry was used to measure daunomycin accumulation in a MDR L. tropica line, a reversing effect was observed with flavones such as dimethylallyl-kaempferide at low concentration or apigenin at higher concentration, but neither with the glucorhamnosyl derivative rutin nor with the isoflavone genistein. The in vivo reversing effect of dimethylallyl-kaempferide was correlated to a high inhibition of MDR cell growth in the presence of daunomycin. The results suggest that flavone inhibition of both daunomycin efflux and parasite growth in the presence of the drug correlates to direct binding of the compound to cytosolic domain of the P-glycoprotein-like transporter.

Characterization of a new ATP-binding cassette transporter in Trypanosoma cruzi associated to a L1Tc retrotransposon.

We have characterized the tcpgp1-like gene of Trypanosoma cruzi, a new ATP-binding cassette (ABC) transporter. tcpgp1 codes for a 1035 amino acid protein with a considerable homology to LtpgpA of Leishmania. Tcpgp1 lacks the conserved sequences corresponding to the second nucleotide-binding domain of other ABC transporters due to the insertion of the L1Tc non-LTR retrotransposon.

Involvement of thiol metabolism in resistance to glucantime in Leishmania tropica.

Clinical resistance to pentavalent antimonials, in the form of pentostam (sodium stibogluconate) or glucantime (N-methylglucamine antimoniate), has long been recognized as a problem in Leishmaniasis. However, the mechanisms of resistance are unclear. We selected in vitro a Leishmania tropica line resistant to 1.2 mg/mL of Sb(V) of glucantime (GLU-R10). The cell line has a stable phenotype for at least 6 months and a resistance index of 1400-fold. The resistant line has no cross-resistance to pentostam or to SbCl3 and SbCl5. The resistance to glucantime was reverted by buthionine sulfoximine (BSO) and chlorambucil (CLB); however, thiol analyses by HPLC of wild-type and GLU-R10 cell lines, in the presence or absence of the drug, showed no differences between these two cell lines. The resistant line had a DNA amplification shown as a circular extrachromosomal element (G-circle) of approximately 22 kb. However, the specific probes for gamma-glutamyl cysteine synthetase, ornithine decarboxylase and trypanothione reductase did not recognize the G-circle amplified in the GLU-R10. The G-circle did not arise from the H region and was not related with P-glycoprotein Pgp-MDR- or Pgp-MRP-like genes. Northern blot analysis of the G-circle showed that a single transcript of approximately 6 kb was overexpressed in the resistant line. Molecular characterization of the G-circle would lead to the determination of the gene(s) involved in resistance to glucantime in Leishmania.

A new member of YER057c family in Trypanosoma cruzi is adjacent to an ABC-transporter.

Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2. The protein contains 160 amino acid residues with a predicted molecular mass of 16.5kDa. Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized. Here, we propose a new signature of this family considering the N-terminal: [IV]-X(4)-[AV]-[AP]-X-[AP]-X(3)-Y-X(9)-[LIVF]-X(2)-[SA]-G-[QS], and the C-terminal: [AT]-R-X(2)-[IVFY]-X-[VC]-X(2)-L-P-X(4)-[LIVM]-E-[IVM] -[DE] motifs. Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles. Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.

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.

A pteridine reductase gene ptr1 contiguous to a P-glycoprotein confers resistance to antifolates in Trypanosoma cruzi.

We have isolated the pteridine reductase-1 gene (ptr1), from Trypanosoma cruzi (Y strain), located contiguous to the Trypanosoma cruzi P-glycoprotein-2 (tcpgp2). The gene encodes a member of the family of short-chain dehydrogenases, enzymes that are involved in several oxidoreduction reactions. One member of the family, pteridine reductase-1 (PTR1) has been previously described in Leishmania as being involved in antifolate resistance. The ptr1 gene from T. cruzi presents an 828 bp open reading frame, coding for a 276 amino acid protein with a predicted molecular mass of 30 kDa. The deduced amino acid sequence exhibited a remarkable homology with the ptr1 genes of Leishmania major and Leishmania tarentolae. Southern blot analysis using a specific probe indicated that T. cruzi PTR1 is encoded by a single copy gene located in two chromosomes of about 0.9 and 1.2 Mb. Western blot analysis using a polyclonal antiserum against recombinant PTR1 revealed that the protein is only expressed in the epimastigote forms of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. Purified recombinant PTR1 exhibits a NADPH-dependent pteridine reductase activity comparable with those described in Leishmania. Gene transfection experiments using the pTEX expression vector show that, under the conditions tested, T. cruzi PTR1 is involved in resistance to the methotrexate, aminopterin and trimethoprim antifolates.

Molecular characterization of a P-glycoprotein-related tcpgp2 gene in Trypanosoma cruzi.

We have cloned, sequenced and characterized a gene from Trypanosoma cruzi (Y strain), termed tcpgp2, which encodes a member of the ABC (ATP-binding cassette) superfamily of evolutionarily conserved transport proteins. The nucleotide sequence of the tcpgp2 gene was determined. It presents a 4602-bp open reading frame, coding for a 1534-amino acid protein, with a predicted molecular mass of 169,470 Da. The deduced amino acid sequence of tcpgp2 exhibited a remarkable homology with the P-glycoprotein-related genes of Leishmania tarentolae, the yeast cadmium factor (YCF1) and the human multidrug resistance-associated protein (MRP). Southern blot analysis using a specific probe indicated that the Tcpgp2 P-glycoprotein is encoded by a single copy gene which maps to a chromosome of about 900 kb. Northern blot analysis revealed that tcpgp2 gene is expressed as a polyadenylated transcript of approximately 5 kb in dividing amastigote and epimastigote forms; we did not detect the transcript in the non-dividing trypomastigote forms of the parasite. Gene transfection experiments in Leishmania tropica indicated that, under the conditions tested, tcpgp2 gene is not involved in drug resistance.