Federico Gago

Regiospecific Synthesis and Anti-Human Immunodeficiency Virus Activity of Novel 5-Substituted N-Alkylcarbamoyl and N,N-Dialkyl Carbamoyl 1,2,3-Triazole-TSAO Analogues

Several 5-N-alkyl and 5-N,N-dialkylcarbamoyl substituted analogues of the anti-human immunodeficiency virus (HIV) type 1 lead compound[1-[2‘,5’-bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-5-(N,N-dimethylcarbamoyl)-1,2,3-triazole]-3‘-spiro-5“-(4”-amino-1“,2”-oxathiole-2“,2”-dioxide) have been prepared and evaluated as inhibitors of HIV-1 replication. A new regiospecific synthetic procedure is described. The compounds were prepared by cycloaddition of the appropriate glycosylazide to 2-oxo-alkylidentriphenyl-phosphoranes, followed by treatment with primary or secondary amines, to yield, exclusively, 5-substituted 1,2,3-triazole-TSAO analogues. Several 5-substituted 1,2,3-triazole-TSAO derivatives proved to be potent inhibitors of HIV-1 replication with higher antiviral selectivity than that of the parent TSAO prototype.

Cross-Talk between Nucleotide Excision and Homologous Recombination DNA Repair Pathways in the Mechanism of Action of Antitumor Trabectedin

Trabectedin (Yondelis) is a potent antitumor drug that has the unique characteristic of killing cells by poisoning the DNA nucleotide excision repair (NER) machinery. The basis for the NER-dependent toxicity has not yet been elucidated but it has been proposed as the major determinant for the drugs cytotoxicity. To study the in vivo mode of action of trabectedin and to explore the role of NER in its cytotoxicity, we used the fission yeast Schizosaccharomyces pombe as a model system. Treatment of S. pombe wild-type cells with trabectedin led to cell cycle delay and activation of the DNA damage checkpoint, indicating that the drug causes DNA damage in vivo. DNA damage induced by the drug is mostly caused by the NER protein, Rad13 (the fission yeast orthologue to human XPG), and is mainly repaired by homologous recombination. By constructing different rad13 mutants, we show that the DNA damage induced by trabectedin depends on a 46-amino acid region of Rad13 that is homologous to a DNA-binding region of human nuclease FEN-1. More specifically, an arginine residue in Rad13 (Arg961), conserved in FEN1 (Arg314), was found to be crucial for the drugs cytotoxicity. These results lead us to propose a model for the action of trabectedin in eukaryotic cells in which the formation of a Rad13/DNA-trabectedin ternary complex, stabilized by Arg961, results in cell death.

The dual role of thymidine phosphorylase in cancer development and chemotherapy.

Thymidine phosphorylase (TP), also known as “platelet‐derived endothelial cell growth factor” (PD‐ECGF), is an enzyme, which is upregulated in a wide variety of solid tumors including breast and colorectal cancers. TP promotes tumor growth and metastasis by preventing apoptosis and inducing angiogenesis. Elevated levels of TP are associated with tumor aggressiveness and poor prognosis. Therefore, TP inhibitors are synthesized in an attempt to prevent tumor angiogenesis and metastasis. TP is also indispensable for the activation of the extensively used 5‐fluorouracil prodrug capecitabine, which is clinically used for the treatment of colon and breast cancer. Clinical trials that combine capecitabine with TP‐inducing therapies (such as taxanes or radiotherapy) suggest that increasing TP expression is an adequate strategy to enhance the antitumoral efficacy of capecitabine. Thus, TP plays a dual role in cancer development and therapy: on the one hand, TP inhibitors can abrogate the tumorigenic and metastatic properties of TP; on the other, TP activity is necessary for the activation of several chemotherapeutic drugs. This duality illustrates the complexity of the role of TP in tumor progression and in the clinical response to fluoropyrimidine‐based chemotherapy.

Mutational Pathways, Resistance Profile, and Side Effects of Cyanovirin Relative to Human Immunodeficiency Virus Type 1 Strains with N-Glycan Deletions in Their gp120 Envelopes

ABSTRACT Limited data are available on the genotypic and phenotypic resistance profile of the α-(1-2)mannose oligomer-specific prokaryotic lectin cyanovirin (CV-N). Therefore, a more systematic investigation was carried out to obtain a better view of the interaction between CV-N and human immunodeficiency virus type 1 (HIV-1) gp120. When HIV-1-infected CEM cell cultures were exposed to CV-N in a dose-escalating manner, a total of eight different amino acid mutations exclusively located at N-glycosylation sites in the envelope surface gp120 were observed. Six of the eight mutations resulted in the deletion of high-mannose type N-glycans (i.e., at amino acid positions 230, 332, 339, 386, 392, and 448). Two mutations (i.e., at position 136 and 160) deleted a complex type N-glycan in the variable V1/V2 domain of gp120. The level of phenotypic resistance of the mutated virus strains against CV-N generally correlated with the number of glycan deletions in gp120, although deletion of the glycans at N-230, N-392, and N-448 generally afforded a more pronounced CV-N resistance than other N-glycan deletions. However, the extent of the decrease of antiviral activity of CV-N against the mutated virus strains was markedly less pronounced than observed for α(1-3)- and α(1-6)-mannose-specific plant lectins Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), which points to the existence of a higher genetic barrier for CV-N. This is in agreement with a more consistent suppression of a wider variety of HIV-1 clades by CV-N than by HHA and GNA. Whereas the antiviral and in vitro antiproliferative activity of CV-N can be efficiently reversed by mannan, the pronounced mitogenic activity of CV-N on peripheral blood mononuclear cells was unaffected by mannan, indicating that some of the observed side effects of CV-N are unrelated to its carbohydrate specificity/activity.

ET-18-OCH3 (Edelfosine): A Selective Antitumour Lipid Targeting Apoptosis Through Intracellular Activation of Fas/CD95 Death Receptor

Synthetic ether-linked analogues of phosphatidylcholine and lysophosphatidylcholine, collectively named as antitumour lipids (ATLs), were initially synthesized in the late 60s, but have attracted a renewed interest since the finding that the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, edelfosine), a synthetic analogue of 2-lysophosphatidylcholine considered the ATL prototype, induces a selective apoptotic response in tumour cells, sparing normal cells. Unlike most chemotherapeutic agents currently used, ET-18-OCH3 does not interact with DNA, but act at the cell membrane, and thereby its effects seem to be independent of the proliferative state of target cells. Each part of the molecular structure of ET-18-OCH3 is important for its optimal proapoptotic activity. Recent progress has unveiled the molecular mechanism underlying the apoptotic action of ET-18-OCH3, involving membrane rafts and Fas/CD95 death receptor, and has led to the proposal of a two-step model for the ET-18-OCH3 selective action on cancer cells, namely: a) ET-18-OCH3 uptake into the tumour cell, but not in normal cells; b) intracellular activation of Fas/CD95 through its translocation and capping into membrane rafts. ET-18-OCH3 constitutes the first antitumour drug acting through the intracellular activation of the Fas/CD95 death receptor. Computational docking studies have allowed us to propose a molecular model for the putative interaction of ET-18-OCH3 with the intracellular Fas/CD95 death domain. This novel mechanism of action represents a new way to target tumour cells in cancer chemotherapy and can be of interest as a new framework in designing novel and more selective proapoptotic antitumour drugs.

Regulation of cyclooxygenase activity by metamizol.

The ability of metamizol to inhibit cyclooxygenase-1 and cyclooxygenase-2 activities has been evaluated using different cyclooxygenase sources. Metamizol inhibited purified cyclooxygenase-1 and cyclooxygenase-2 with an IC50 of about 150 microg/ml. A similar IC50 value for cyclooxygenase-2 was obtained in lipopolysaccharide-activated broken murine macrophages. Consistent with these findings, molecular models of the complexes between cyclooxygenase-1 or cyclooxygenase-2 with 4-methylaminoantipyrine, the major active derivative of metamizol, suggested a common binding mode to both isoforms. In intact cells, however, the inhibition profiles were markedly different. The IC50 values of metamizol for cyclooxygenase-1 in intact bovine aortic endothelial cells (BAEC) cells and human platelets were 1730 +/- 150 microg/ml and 486 +/- 56 microg/ml, respectively. Inhibition of cyclooxygenase-2 activity in murine macrophages and primary human leukocytes activated by lipopolysaccharide yielded IC50 values of 12 +/- 1.8 microg/ml and 21 +/- 2.9 microg/ml, respectively. These data indicate that the IC50 values obtained with purified enzymes or disrupted cells cannot always be extrapolated to the cyclooxygenase inhibitory activity of nonsteroidal antiinflammatory drugs (NSAIDs) in intact cells. The data presented here also indicate that cyclooxygenase-2 inhibition could play an important role in the pharmacological effects of metamizol.

3D-QSAR methods on the basis of ligand-receptor complexes. Application of COMBINE and GRID/GOLPE methodologies to a series of CYP1A2 ligands.

Many heterocyclic amines (HCA) present in cooked food exert a genotoxic activity when they are metabolised (N-oxidated) by the human cytochrome P450 1A2 (CYP1A2h). In order to rationalize the observed differences in activity of this enzyme on a series of 12 HCA, 3D-QSAR methods were applied on the basis of models of HCA–CYP1A2h complexes. The CYP1A2h enzyme model has been previously reported and was built by homology modeling based on cytochrome P450 BM3. The complexes were automatically generated applying the AUTODOCK software and refined using AMBER. A COMBINE analysis on the complexes identified the most important enzyme–ligand interactions that account for the differences in activity within the series. A GRID/GOLPE analysis was then performed on just the ligands, in the conformations and orientations found in the modeled complexes. The results from both methods were concordant and confirmed the advantages of incorporating structural information from series of ligand–receptor complexes into 3D-QSAR methodologies.

Molecular pharmacology and antitumor activity of Zalypsis® in several human cancer cell lines

Zalypsis is a new synthetic alkaloid tetrahydroisoquinoline antibiotic that has a reactive carbinolamine group. This functionality can lead to the formation of a covalent bond with the amino group of selected guanines in the DNA double helix, both in the absence and in the presence of methylated cytosines. The resulting complex is additionally stabilized by the establishment of one or more hydrogen bonds with adjacent nucleotides in the opposite strand as well as by van der Waals interactions within the minor groove. Fluorescence-based thermal denaturation experiments demonstrated that the most favorable DNA triplets for covalent adduct formation are AGG, GGC, AGC, CGG and TGG, and these preferences could be rationalized on the basis of molecular modeling results. Zalypsis-DNA adducts eventually give rise to double-strand breaks, triggering S-phase accumulation and apoptotic cell death. The potent cytotoxic activity of Zalypsis was ascertained in a 24 cell line panel. The mean IC(50) value was 7nM and leukemia and stomach tumor cell lines were amongst the most sensitive. Zalypsis administration in four murine xenograft models of human cancer demonstrates significant tumor growth inhibition that is highest in the Hs746t gastric cancer cell line with no weight loss of treated animals. Taken together, these results indicate that the potent antitumor activity of Zalypsis supports its current development in the clinic as an anticancer agent.

High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations

ABSTRACT To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs.

HIV Protease Inhibition: Limited Recent Progress and Advances in Understanding Current Pitfalls

The identification of HIV-1 protease (HIVp) as a target for therapeutic intervention against AIDS was soon followed by major efforts to understand its substrate specificity, reaction kinetics and three-dimensional structure, both in the free state and in complex with a number of ligands including substrate mimics, products, and inhibitors. On the whole these studies have been extremely successful and have had a major impact on our understanding of ligand-receptor interactions and enzyme inhibition mechanisms. HIVp has also become a paradigm for the development and testing of new drug-design methodologies both in vitro and in silico. Even though thousands of potential HIVp inhibitors exhibiting amazing chemical diversity have been synthesized or identified from natural sources, only a few have turned out to be useful for human therapy. Although the alternative goal of preventing enzyme dimerization has been achieved as a proof of concept, this approach has not yet yielded a clinical candidate. The review covers the general strategies that led to some of the most useful inhibitors, the reasons for our limited success in effectively inhibiting this retroviral target in a clinical setting, current progress with second-generation inhibitors, and new avenues for research.