Adolfo M. Iribarren

Inhibition of in vivo breast cancer growth by antisense oligodeoxynucleotides to type I insulin-like growth factor receptor mRNA involves inactivation of ErbBs, PI-3K/Akt and p42/p44 MAPK signaling pathways but not modulation of progesterone receptor activity.

The present study addresses the effect of targeting type I insulin-like growth factor receptor (IGF-IR) with antisense strategies in in vivo growth of breast cancer cells. Our research was carried out on C4HD tumors from an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice. We employed two different experimental strategies. With the first one we demonstrated that direct intratumor injection of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to IGF-IR mRNA resulted in a significant inhibition of C4HD tumor growth. In the second experimental strategy, we assessed the effect of intravenous (i.v.) injection of AS [S]ODN on C4HD tumor growth. This systemic treatment also resulted in significant reduction in tumor growth. The antitumor effect of IGF-IR AS[S]ODNs in both experimental protocols was due to a specific antisense mechanism, since growth inhibition was dose-dependent and no abrogation of tumor proliferation was observed in mice treated with phosphorothioate sense ODNs (S[S]ODNs). In addition, IGF-IR expression was inhibited in tumors from mice receiving AS[S]ODNs, as compared to tumors from control groups. We then investigated signal transduction pathways modulated in vivo by AS[S]ODNs treatment. Tumors from AS[S]ODN-treated mice of both intratumoral and intravenous protocols showed a significant decrease in the degree of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation. Activation of two of the main IGF-IR signaling pathways, phosphatidylinositol 3-kinase (PI-3K)/Akt and p42/p44 mitogen-activated protein kinases (MAPK) was abolished in tumors growing in AS[S]ODN-treated animals. Moreover, ErbB-2 tyrosine phosphorylation was blocked by in vivo administration of AS[S]ODNs. On the other hand, we found no regulation of either progesterone receptor expression or activity by in vivo AS[S]ODNs administration. Our results for the first time demonstrated that breast cancer growth can be inhibited by direct in vivo administration of IGF-IR AS[S]ODNs.

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

Nucleosides are valuable bioactive molecules, which display antiviral and antitumour activities. Diverse types of prodrugs are designed to enhance their therapeutic efficacy, however this strategy faces the troublesome selectivity issues of nucleoside chemistry. In this context, the aim of this review is to give an overview of the opportunities provided by biocatalytic procedures in the preparation of nucleoside prodrugs. The potential of biocatalysis in this research area will be presented through examples covering the different types of nucleoside prodrugs: nucleoside analogues as prodrugs, nucleoside lipophilic prodrugs and nucleoside hydrophilic prodrugs.

Complete and regioselective deacetylation of peracetylated uridines using a lipase

Lipase-catalysed alcoholysis and hydrolysis of 2′,3′,5′-tri-O-acetyluridine (1a) and 2′,3′,5′-tri-O-acetyl-2′-C-methyluridine (1b) were studied. Conditions for full and regioselective deacetylation of 1aand 1b are shown in the present work. New compound 2′,3′-di-O-acetyl-2′-C-methyluridine (3b) was prepared by regioselective lipase-catalysed deacetylation.

Purine nucleoside synthesis from uridine using immobilised Enterobacter gergoviae CECT 875 whole cells

Abstract Biocatalysed purine nucleoside synthesis was carried out using immobilised Enterobacter gergoviae CECT 875. Similar yields (80–95%) in adenosine were obtained with both free and immobilised cells though in the last case a long reaction time was necessary. The immobilised cells can be reused at least for more than 30 times without significant loss of enzymatic activity. The immobilised biocatalyst in agarose is active in the synthesis of unnatural nucleosides.

Modified Nucleoside Triphosphates for In-vitro Selection Techniques

The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed.

Screening of catalytically active microorganisms for the synthesis of 6-modified purine nucleosides.

Modified nucleosides can be prepared by microbial transglycosylation from cheaper nucleoside precursors using free or immobilised whole cells. An efficient screening method to find transglycosylation activity in␣microorganisms was developed for the synthesis of 6-modified purine nucleosides, such as 6-chloro-, 6-methoxy-, 6-iodo- and 6-mercaptopurine ribonucleoside. Out of 100 microorganisms screened, Bacillus stearothermophilus ATCC 12980 was the best for this purpose.

Regioselective preparation of 2′, 3′-di-O-acylribonucleosides carrying lipophilic acyl groups through a lipase-catalysed alcoholysis

Candida antarctica B lipase-catalysed alcoholysis of 2′, 3′, 5′-tri-O-hexanoyluridine (1a), 2′, 3′, 5′-tri-O-dodecanoyluridine (1b), 2′, 3′, 5′-tri-O-hexanoylinosine (1c) and 2′, 3′, 5′-tri-O-dodecanoylinosine (1d) proceeded regioselectively to produce the corresponding 2′, 3′-di-O-acylribonucleosides 2a–d, providing a simple and efficient access to these new lipophilic compounds. Contrasting to the alcoholysis, enzymatic hydrolysis of 1a–d using different enzymes and experimental conditions did not proceed regioselectively.

A simple and efficient enzymatic procedure for the deprotection of two base labile chlorinated purine ribosides

Base-labile 6-chloro-2′,3′,5′-tri-O-acetylpurine riboside (1c) and 2-amino-6-chloro-2′,3′,5′-tri-O-acetylpurine riboside (1d) were fully deacetylated through Candida antarctica B lipase hydrolysis, affording respectively 6-chloropurine riboside (2c) and 2-amino-6-chloro-purine riboside (2d). Quantitative results were found at pH 7 and 60 °C in 24 h for 1c and 72 h for 1d. This mild and simple enzymatic technique represents a convenient procedure for the removal of acetyl groups from such base labile halogenated nucleosides.

Microbial Hydrolysis of Acetylated Nucleosides

Enzymatic hydrolysis of acetylated nucleosides using microbial whole cells has been carried out for the first time. Unlike Candida antarctica B lipase-catalysed alcoholysis, none of the tested microorganisms displayed a common deacetylation profile. Depending on the substrate and the biocatalyst used, 5′-selective deprotection or mixtures of mono O-acetylated products were obtained.

Activity of core-modified 10-23 DNAzymes against HCV

The highly conserved untranslated regions of the hepatitis C virus (HCV) play a fundamental role in viral translation and replication and are therefore attractive targets for drug development. A set of modified DNAzymes carrying (2′R)‐, (2′S)‐2′‐deoxy‐2′‐C‐methyl‐ and ‐2′‐O‐methylnucleosides at various positions of the catalytic core were assayed against the 5′‐internal ribosome entry site element (5′‐IRES) region of HCV. Intracellular stability studies showed that the highest stabilization effects were obtained when the DNAzymes′ cores were jointly modified with 2′‐C‐methyl‐ and 2′‐O‐methylnucleosides, yielding an increase by up to fivefold in the total DNAzyme accumulation within the cell milieu within 48 h of transfection. Different regions of the HCV IRES were explored with unmodified 10–23 DNAzymes for accessibility. A subset of these positions was tested for DNAzyme activity using an HCV IRES‐firefly luciferase translation‐dependent RNA (IRES‐FLuc) transcript, in the rabbit reticulocyte lysate system and in the Huh‐7 human hepatocarcinoma cell line. Inhibition of IRES‐dependent translation by up to 65 % was observed for DNAzymes targeting its 285 position, and it was also shown that the modified DNAzymes are as active as the unmodified one.