Biochemical and structural characterization of the RT domain of Leishmania sp. telomerase reverse transcriptase.

BACKGROUND: The Leishmania genus comprises parasites that cause leishmaniasis, a neglected disease spread worldwide. Leishmania sp. telomeres are composed of TTAGGG repeats maintained by telomerase. In most eukaryotes, the enzyme minimal complex contains the TER (telomerase RNA) and the TERT (telomerase reverse transcriptase) components. The TERT holds the enzyme catalytic core and is formed by four structural and functional domains (TEN, Telomerase Essential N-terminal; TRBD, Telomerase RNA Binding Domain; RT, the reverse transcriptase domain and CTE, C-Terminal Extension domain). METHODS AND RESULTS: Amino acid sequence alignments, protein structure prediction analysis, and protein: nucleic acid interaction assays were used to show that the Leishmania major RT domain preserves the canonical structural elements found in higher eukaryotes, including the canonical motifs and the aspartic acid residues that stabilize the Mg2+ ion cofactor. Furthermore, amino acid substitutions specific to the Leishmania genus and partial conservation of the residues involved with nucleic acid interactions are shown. The purified recombinant Leishmania RT protein is biochemically active and interacts with the G-rich telomeric strand and the TER template sequence. CONCLUSION: Our results highlight that the telomerase catalysis mechanism is conserved in a pathogen of medical importance despite the structural peculiarities present in the parasite's RT domain.

Homology Model and Docking-Based Virtual Screening for Ligands of Human Dyskerin as New Inhibitors of Telomerase for Cancer Treatment.

Immortality is one of the main features of cancer cells. Tumor cells have an unlimited replicative potential, principally due to the holoenzyme telomerase. Telomerase is composed mainly by dyskerin (DKC1), a catalytic retrotranscriptase (hTERT) and an RNA template (hTR). The aim of this work is to develop new inhibitors of telomerase, selecting the interaction between hTR⁻DKC1 as a target. We designed two models of the human protein DKC1: homology and ab initio. These models were evaluated by different procedures, revealing that the homology model parameters were the most accurate. We selected two hydrophobic pockets contained in the PUA (pseudouridine synthase and archaeosine transglycosylase) domain, using structural and stability analysis. We carried out a docking-based virtual screen on these pockets, using the reported mutation K314 as the center of the docking. The hDKC1 model was tested against a library of 450,000 drug-like molecules. We selected the first 10 molecules that showed the highest affinity values to test their inhibitory activity on the cell line MDA MB 231 (Monroe Dunaway Anderson Metastasis Breast cancer 231), obtaining three compounds that showed inhibitory effect. These results allowed us to validate our design and set the basis to continue with the study of telomerase inhibitors for cancer treatment.

Human telomerase protein: Understanding how the catalytic activity is suppressed under single substitutions of some conserved residues. A computational study.

The reverse transcriptase domain in telomerase proteins contains the essential conserved residues to catalyze the addition of a single nucleotide to the ends of DNA strands of most eukaryotic cells. In human telomerase protein, mutations in the conserved residues K902, R631, K626, D712, D868, and D869 are known to suppress catalytic activity. To understand these results, a computational model was constructed to simulate a ternary complex consisting of a model of the protein reverse transcriptase domain, a DNA/RNA double helix, an incoming dNTP, and two Mg2+ ions. Three independent Molecular Dynamics Simulations were performed for the wild type and the mutated K902N, R631Q, D712A, D868A, and D869A complexes. Binding Free Energies and alanine-scanning studies were also performed. Using the two-metal-ion mechanism for the nucleotide addition, deviations from the wild type which stop the activity of the human protein, were identified in each mutated complex. The K902N and R631Q mutations might stop the catalytic activity preventing the exit of the pyrophosphate from the catalytic pocket. Additionally, evidence that the same mechanism probably applies to the K626A, R631A, and K902A mutations is presented. For D712A mutation, the pentavalent intermediate state might not form; therefore, the catalytic reaction might not even begin. For the D868A mutation, the O3'-hydroxyl might lose coordination with the Mg ion and the reaction might not either start. Finally, from the limited sampling carried out in this work, we did not obtain any evidence to identify the mechanism by which the D869A mutation cancels the activity of telomerase.

FDA-approved drugs selected using virtual screening bind specifically to G-quadruplex DNA.

Guanine-rich sequences found in telomeres and oncogene promoters have the ability to form G-quadruplex structures. In this paper we describe the use of a virtual screening assay to search a database of FDA-approved compounds for compounds with the potential to bind G-quadruplex DNA. More than 750 telomerase inhibitors were identified in a literature search as acting through G-quadruplex stabilization, and from evaluation of these compounds, theoretical models capable of discriminating new compounds that bind G-quadruplex DNA were developed. Six compounds predicted to bind to the G-quadruplex structure were tested for their ability to bind to the human telomeric DNA sequence. Prochloroperazine, promazine, and chlorpromazine stabilized the G-quadruplex structure as determined by fluorescence resonance energy transfer techniques. These compounds also bound to promoter sequences of oncogenes such as c-myc and K-ras. Amitriptyline, imipramine, and loxapine were less stabilizing but did bind to the G-quadruplex. The ability of prochloroperazine, promazine, and chlorpromazine to recognize G-quadruplex structures was confirmed using a fluorescent intercalator displacement assay, in which displacement of thiazole orange from G-quadruplex structures was demonstrated. Interestingly, these compounds exhibited selectivity for the G-quadruplex structure as all had poor affinity for the duplex sequence.

Defects in mTR stability and telomerase activity produced by the Dkc1 A353V mutation in dyskeratosis congenita are rescued by a peptide from the dyskerin TruB domain.

BACKGROUND: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. MATERIALS AND METHODS: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. RESULTS: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. DISCUSSION: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.

Computational tools in the discovery of new G-quadruplex ligands with potential anticancer activity.

Guanine-rich sequences found at telomeres and oncogenes have the capacity to form G-quadruplex (G4) structures. It has been found a relationship between the ability to stabilizing G4 structures and anticancer activity. Guanine quadruplexes stabilization and its implication in cancer phenomena is a therapeutic target relatively recent. Computer-aided drug design has been a very useful tool for the search of new candidates. In last years, methodologies have improved with the development of the computational sciences. The hardware is also enhanced, new techniques are explored. NMR and X-ray information about different targets are discovered continually. The continuous augmentation of new powerful and comprehensive software's with this purpose is other significant factor that contributes to the discovering of new compounds. Nevertheless computer-aided drug design has not been vastly employed in the design of new compound with G4 stabilization activity. All things considered, this review will be focused on the influence of computational techniques on speeding up the discovery of new G4 ligands.

Characterization of Trypanosoma cruzi telomerase.

High telomerase activity is always associated with actively dividing cells, however the detection of this activity in dividing Leishmania and Trypanosoma cruzi cells has always been disappointingly low. Recently, we have found that Leishmania major telomerase activity can be activated by heat, which combined with dilutions of the nuclear extracts produced an increase in activity comparable to cancer cells. Here we examined whether T. cruzi telomerase shares the same physicochemical properties of primer specificity and overall features of the L. major. Our studies revealed that no telomerase inhibitory factors were present in the nuclear lysates of T. cruzi however the enzyme was activated by heat and was very resilient to heat denaturation. We also showed the extension primer specificity, susceptibility to RNase-A and RNase-H digestion, and the effect of telomerase inhibitors.

The putative telomerase reverse transcriptase component of Leishmania amazonensis: gene cloning and characterization.

The Leishmania amazonensis telomerase gene was cloned by a polymerase chain reaction-based strategy using primers designed from a Leishmania major sequence that shared similarities with conserved telomerase motifs. The genes from three other species were cloned for comparative purposes. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania telomerase gene was probably in single copy and located in the largest chromosomes. A single messenger ribonucleic acid transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases.

[Telomerase: an enzyme with multiple applications in cancer research]. / La telomerasa: una enzima con múltiples aplicaciones en la investigación del cáncer.

The telomerase is a ribonucleoprotein enzyme to which multiple functions have been attributed, the most important of these is the maintenance of the telomere which is related with cellular immortalization and cancer. 85% of human tumors have telomerase activity, that in normal cells goes undetected. These characteristics make the telomerase an attractive target for chemotherapy.