Clemastine/tamoxifen hybrids as easily accessible antileishmanial drug leads.

A library of hybrid molecules is developed based on the common chemical features shared by clemastine and tamoxifen both of which are well known for their antileishmanial activities. In the initial screening against Leishmania major and L. amazonensis promastigotes, as well as cytotoxicity assays using HepG2 cells, several hybrids showed submicromolar activity against the parasite and no toxicity against human cells. The compounds with an EC50 < 2 µM against promastigotes of both species and a selectivity index >10 were further characterized against intracellular amastigotes as well as promastigotes of species that cause both visceral and cutaneous leishmaniasis, such as L. infantum and L. braziliensis, respectively. These sequential screenings revealed the high pan-activity of this class of molecules against these species, with several compounds displaying an EC50 ≤ 2 µM against both promastigotes and intracellular amastigotes. Two of them were identified as the potential templates for lead optimization of this series having shown the highest activities against all species in both stages of parasite. The present findings can serve as a good starting point in the search for novel antileishmanial compounds that are easy to access and highly active.

Leishmania: overexpression and comparative structural analysis of the stage-regulated meta 1 gene.

The meta 1 gene of Leishmania major is upregulated in metacyclic promastigotes and encodes an 11.5-kDa protein with no significant similarities to other proteins in the existing databases. In this paper, we characterize the homologous meta 1 genes in L. amazonensis and L. donovani. Proteins encoded by this gene in all three species present a high degree of identity. The meta 1 gene cannot be replaced by gene targeting in L. major, suggesting an essential role for the protein, at least in promastigotes. Overexpression of the meta 1 protein in L. amazonensis generates parasites that are more virulent than wild-type organisms in vivo.

Characterisation and expression of a stage-regulated gene of Leishmania major.

This paper describes the isolation and characterisation of the meta 1 gene from Leishmania major, that is expressed predominantly in infective metacyclic parasites. The termini of the major 2-kb transcript derived from this gene have been mapped and polypyrimidine sequences implicated in RNA processing located in the flanking regions. The meta 1 gene is conserved in both Old and New World Leishmania species and codes for a putative protein of 112 amino acids. Antibodies raised against a recombinant protein expressed from this open reading frame recognise an 11.5-kDa protein in metacyclic cell lysates and this molecule localises to the region of the flagellar pocket by indirect immunofluorescence.

Structural and functional characterization of the Leishmania amazonensis ribosomal RNA promoter.

The promoter region of the ribosomal RNA (rRNA) genes of Leishmania amazonensis was characterised and the transcription start point, defined by primer extension, was shown to be a T residue, 1048 nucleotides upstream of the beginning of the 18S sequence. A repetitive element of 60 bp was identified in the intergenic spacer. This element did not show sequence similarity with the region around the transcription start point. Conserved sequences were found in the external transcribed spacer of L. amazonensis, Trypanosoma cruzi and Crithidia fasciculata rRNA genes, 150 nucleotides downstream of the transcription start point. These sequences might be involved in processing events of the rRNA precursor molecule. The general organisation of the gene resembles the pattern observed for the ribosomal cistron in eukaryotic cells. Constructs containing the L. amazonensis promoter region upstream of the chloramphenicol acetyltransferase (cat) gene were able to drive the expression of the reporter gene in transient transfection experiments. CAT expression could be detected even when no trans-splicing acceptor sequence was added to the constructs, although its presence enhanced 5-fold the level of CAT activity. Species-specificity of the RNA polymerase I promoter activity was also demonstrated since constructs containing the L. amazonensis promoter region were unable to drive CAT expression when transfected into the related trypanosomatids, T. cruzi, C. fasciculata and Endotrypanum schaudini.

Discrimination amongst Leishmania by polymerase chain reaction and hybridization with small subunit ribosomal DNA derived oligonucleotides.

A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.

Optimization of coupled PCR amplification and cycle sequencing of cloned and genomic DNA.

We describe optimization of a coupled amplification and cycle sequencing (CAS) method for rapid characterization of cloned or genomic DNA. Our modification of this method, termed coupled PCR amplification and cycle sequencing (CPACS), utilizes commercially available reagents, does not require template purification and produces high-quality sequence ladders from nanogram quantities of complex genomic DNA. The reactions have been streamlined to permit automation. Finally, we show that the technique can be applied more efficiently in conjunction with the AutoTrans 350 Direct Transfer Electrophoresis System and 33P-labeled sequencing primers.

Ribosomal DNA restriction analysis and synthetic oligonucleotide probing in the identification of genera of lower trypanosomatids.

Fifty-four species or isolates of insect trypanosomatids were examined for the presence of selected restriction enzyme sites in the small (SSU) and large (LSU) rRNA coding units of ribosomal genes. In the SSU, sites for Eco RI, Bgl II, Pst I, and Hind III were found to occur at the same location for all species examined, thus displaying a universal distribution among trypanosomatids. In the LSU, a site for Bgl II in the 24S-alpha sequence and sites for Hind III and Pst I in the 24S-beta sequence were found in all species examined. In contrast, a site for Pvu II in the SSU exhibited a genus-related distribution, being present in Crithidia and Herpetomonas but absent in Phytomonas. A site for Hind III in the 24S-alpha sequence of the LSU also exhibited genus-restricted distribution. The site was present in Crithidia but absent in Phytomonas and Herpetomonas. These findings were confirmed by dot hybridization with a synthetic oligonucleotide complementary to the 18S rRNA sequence containing the Pvu II site. Results point to the usefulness of restriction markers as diagnostic tools for distinguishing the lower trypanosomatid genera Crithidia, Herpetomonas, and Phytomonas at the same time revealing a marked complexity within the genus Leptomonas.

Leishmania: genus identification based on a specific sequence of the 18S ribosomal RNA sequence.

The analysis of PvuII restriction patterns of Leishmania spp. and Trypanosoma spp. genomic DNA showed genus distinctive profiles. A specific PvuII site was detected in the 5' domain of 18S ribosomal DNA of Leishmania. A 20-mer oligonucleotide encompassing this PvuII region was synthesized. This sequence, when utilized as probe, on short exposures of dot tests, detected 10(3) whole promastigotes of all Leishmania species analyzed but did not hybridize with T. cruzi or human nucleic acids. Two other oligonucleotides were synthesized to be used as primers for amplification through polymerase chain reaction of the 18S ribosomal DNA region containing the PvuII site. The probes described may be useful for the detection of Leishmania spp. under clinical and epidemiological trials.