Current Pharmaceutical Design - Volume 23, Issue 45, 2017

Nucleosides constitute an extensive group of natural and chemically modified compounds that display a wide range of structures and activities. Different biocatalysts have been developed for their preparation, but the choice of commercially available enzymes is limited. Therefore, the search of new biocatalysts is particularly attractive. In this sense, microorganisms are a vast source of enzymatic diversity that can be directly used as whole cell biocatalysts providing a potential cheaper and suitable route for industrial applications. Methods: This work makes particular emphasis on the following methods: the biocatalyzed whole cell synthesis of nucleosides mediated by phosphorylases, key biocatalyzed steps involved in other chemoenzymatic routes to prepare nucleoside analogues and the transformation of nucleosides in derivatives with particular properties. Results: The literature covered in this work confirms that biocatalytic procedures that make use of whole cell systems can be successfully applied to obtain a wide variety of nucleoside analogues and their derivatives, providing alternative and complementary routes to traditional chemistry. The direct use of microbial whole cells as biocatalysts affords competitive results since it avoids the cumbersome procedures involved in enzyme isolation and facilitates multienzymatic processes. These biocatalysts also maintain the enzymes in their natural environment, protecting their activities from reaction conditions. Conclusion: Although the information presented herein shows that these methodologies have reached a high degree of development, it is expected that future contributions of protein engineering and nucleoside metabolism knowledge, among other disciplines, will expand the already wide range of applications in nucleoside chemistry of whole cell biocatalysis.

Background: Nucleoside analogue (NA) derivatives comprise a large family of pharmaceuticals clinically used as antitumoral and antiviral compounds. Originally, the production of NAs involved chemical synthesis, but a greener bioproduction alternative exists and involves the use of enzymes that catalyze transglycosylation reactions between modified purinic or pyrimidinic bases and sugars. To be considered as an option for industrial application, it is vital to immobilize these biocatalysts. Methods: This article describes current methodologies for whole cell and protein immobilization mostly applied to the synthesis of important NAs. Immobilization describes ways of cell or enzyme confinement in diverse surfaces or matrixes. It is important to be familiar with the variety of matrixes and supports available prior to biocatalyst immobilization so the most adequate can be selected for the purpose sought. Results: From the different articles compiled, it can be acknowledged that the main methods for protein or cell stabilization are immobilization by adsorption, covalent, cross-linking and entrapment. The most widely used matrixes and supports are agar, alginate, polyacrylamide, sepharose derivatives, and acrylic resins, among others. Protein or cell stabilization has the advantage of stabilizing immobilization, favoring their facile separation from the reaction medium for further reuse and also making the purification of the final product easier. Moreover, biocatalyst stabilization allows a facile estimation of the economic cost of the bioprocess and of an eventual scale- up, being a basic requirement for industrial application. Conclusion: In order to achieve successful biocatalyst immobilization, parameters such as biocatalyst stability, mechanical resistance, and reusability should be considered. This review describes and summarizes the methods used for the immobilization of biocatalysts for the synthesis of NAs in the last years.

Background: In recent years, enzymatic methods have shown to be an efficient and sustainable alternative for the synthesis of nucleosides and nucleoside-5'-monophosphates (NMPs) to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection–deprotection steps and the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Results: In this mini-review, we want to illustrate the application of phosphoribosyltransferases (PRTs) in enzymatic synthesis of NMPs. In this sense, many different examples about the use of PRTs as biocatalysts, as whole cells or enzymes, are described. In addition, it also includes detailed comments about structure and catalytic mechanism of described PRTs, as well as their possible biological role and therapeutic use, substrate specificity and advances in detection of new enzyme specificities towards different substrates. In addition, several examples about the use of PRTs in mono or multi-enzymatic synthesis of NMP analogues are shown. Finally, a brief discussion about advantages and drawbacks of the use of PRTs as industrial biocatalyst of NMPs has been commented. Conclusion: Despite the great potential of PRTs as biocatalysts for industrial synthesis of NMPs, several drawbacks must be overcome before reaching a suitable industrial application. In this sense, multi-enzyme systems provide an appropriate framework for this purpose. Moreover, future advances in different disciplines as protein engineering, bioinformatics and -omics will help to reach this goal.

Background: Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are relevant to the appropriate function of the immune system in mammals and interesting drug targets for cancer treatment. Next to their role as drug targets nucleoside phosphorylases are used as catalysts in the synthesis of nucleosides and their analogs that are widely applied as pharmaceuticals. Methods: Based on their substrates nucleoside phosphorylases are classified as pyrimidine and purine nucleoside phosphorylases. This article describes the substrate spectra of nucleoside phosphorylases and structural properties that influence their activity. Substrate ranges are summarized and relations between members of pyrimidine or purine nucleoside phosphorylases are elucidated. Results: Nucleoside phosphorylases accept a broad spectrum of substrates: they accept both base and sugar modified nucleosides. The most widely studied nucleoside phosphorylases are those of Escherichia coli, mammals and pathogens. However, recently the attention has been shifted to thermophilic nucleoside phosphorylases due to several advantages. Nucleoside phosphorylases have been applied to produce drugs like ribavirin or fludarabine. However, limitations were observed when drugs show an open ring structure. Site-directed mutagenesis approaches were shown to alter the substrate specificity of nucleoside phosphorylases. Conclusion: Nucleoside phosphorylases are valuable tools to produce modified nucleosides with therapeutic or diagnostic potential with high affinity and specificity. A wide variety of nucleoside phosphorylases are available in nature which differ in their protein sequence and show varying substrate spectra. To overcome limitations of the naturally occurring enzymes site-directed mutagenesis approaches can be used.

Background: Nucleoside 5'-triphosphates (NTPs) play an important role in cells in the transfer of phosphate groups or bioenergy. In vivo, they are ready to be produced, regenerated and consumed in different kinds of metabolic pathways, and their concentrations are strictly controlled. NTPs are useful reagents that take part in many biosynthetic processes. However, NTPs are expensive and unstable, which greatly increases the cost of the final product if a large amount of NTPs is used directly in biosynthesis. Furthermore, during reactions, NTPs degrade into NDPs and need to be separated from the reaction mixture, making the operation complicated. Therefore, NTPs are normally regenerated from NDPs, and only very few NTPs are used in the reaction. Method: Mechanisms of NTP regeneration were analysed, and their applications in the biosynthesis of nucleotides and their derivates were described. Basically, NTP regeneration involves isolated enzyme systems and whole-cell systems. Result: As one type of cofactor regeneration, NTPs can be effectively regenerated by acetate kinase, pyruvate kinase, and polyphosphate kinase from acetyl phosphate, phosphoenol pyruvate, and polyphosphate, respectively, or by whole cells of yeast and Corynebacterium ammoniagenes from simple carbohydrates and phosphate. The NTP-regeneration method is selected primarily due to the main reaction that it is being coupled with. The cost of phosphate donors and the convenience of integration with the main process should be considered. Conclusion: Significant advances have been made when NTP regeneration is coupled with other biosynthetic processes, especially in the preparation of nucleotides, 2'-deoxynucleotides, sugar-nucleotides and their derivatives.

Background: Purine-nucleoside phosphorylase (PNP) is known as a tool for the synthesis of various nucleosides and nucleoside analogues. Mechanism, properties, molecular diversity and inhibitors of PNP, particularly these of pharmacological significance, are briefly characterized. Methods: UV and fluorescence spectroscopy was used for kinetic experiments, and HPLC chromatography for product analyses. Results: Applications of various forms of PNP to synthesis of selected fluorescent nucleosides, particularly ribosides of 1,N6-ethenoadenine and various 8-azapurines (triazolo[4,5-d]pyrimidines) are reviewed. Different specificity of various PNP forms is described towards nucleobase and analog substrates as well as variable ribosylation sites observed in some reactions, with a possibility to further modify these features via the site-directed mutagenesis. Conclusion: Present and future applications of the fluorescent or fluorogenic ribosides are discussed, with particular emphasis on biochemical and clinical analyses with improved sensitivity.

Background: Schistosoma mansoni is the etiological agent of schistosomiasis, a debilitating treatment neglected tropical disease that affects approximately 218 million people worldwide. Despite its importance, the treatment of schistosomiasis relies on a single drug, praziquantel. Some reports on the resistance of S. mansoni to this drug have stimulated efforts to develop new drugs to treat this disease. S. mansoni possesses all the same pyrimidine pathways (de novo, salvage and thymidylate cycles) as those of its host. The opposite scenario is true for purine metabolism, in which only the salvage pathway is present. These pathways have previously been proposed as potential drug targets. Results: Using modern molecular biology techniques, large-scale study of these pathways has become possible; 24 genes have been studied, and several protein structures and kinetic parameters have been determined. Unique characteristics of schistosomal enzymes have been obtained, which show that this organism possesses two isoforms of uridine phosphorylase (UP), which share 92% of identity. However, only one isoform has a canonical function, whereas the second isoform is expressed through all life stages and does not have a known function. In addition, the methylthioadenosine phosphorylase (MTAP) is one of the enzymes responsible for the previously described adenosine phosphorylase activity, thus representing one main difference between S. mansoni and its host. The study of adenine phosphoribosyltransferase has revealed possible differential expression of the APRT gene in females. This result is consistent with those obtained for the experimental treatment of schistosomiasis in monkeys with the adenosine analog tubercidin, which eliminates the disease mainly in females. Conclusion: These important conclusions may aid in the development of new alternative drugs to treat schistosomiasis.

Background: Modified nucleoside and nucleotide analogs are now the cornerstone of antiviral and anticancer chemotherapies. However, these compounds are not active on their own and need, after entering the cell, to be metabolized to their active 5'-triphosphate form. Methods: Limitations of these metabolic processes led to development of nucleoside/nucleotide prodrugs in which nucleosides are masked with different groups that can be intracellularly cleaved either chemically or enzymatically. Results: Several prodrug approaches have been successfully developed in order to increase the efficacy, bioavailability, penetration in target organ, and selectivity of nucleoside/nucleotide analogs. Conclusion: The concept of nucleoside/nucleotide prodrug is now a well-established approach that led to the approval of numerous drugs for the treatment of HIV, HBV, HCV, HSV and cancer.

Background: The selective expression of non-human genes in tumor tissue to activate non-toxic compounds (Gene Directed Enzyme Prodrug Therapy, GDEPT) is a novel strategy designed for killing tumor cells in patients with little or no systemic toxicity. Numerous non-human genes have been evaluated, but none have yet been successful in the clinic. Methods: Unlike human purine nucleoside phosphorylase (PNP), E. coli PNP accepts adenine containing nucleosides as substrates, and is therefore able to selectively activate non-toxic purine analogs in tumor tissue. Various in vitro and in vivo assays have been utilized to evaluate E. coli PNP as a potential activating enzyme. Results: We and others have demonstrated excellent in vitro and in vivo anti-tumor activity with various GDEPT strategies utilizing E. coli PNP to activate purine nucleoside analogs. A phase I clinical trial utilizing recombinant adenoviral vector for delivery of E. coli PNP to solid tumors followed by systemic administration of fludarabine phosphate (NCT01310179; IND# 14271) has recently been completed. In this trial, significant anti-tumor activity was demonstrated with negligible toxicity related to the therapy. The mechanism of cell kill (inhibition of RNA and protein synthesis) is distinct from all currently used anticancer drugs and all experimental compounds under development. The approach has demonstrated excellent ability to kill neighboring tumor cells that do not express E. coli PNP, is active against non-proliferating and proliferating tumors cells (as well as tumor stem cells, stroma), and is therefore very effective against solid tumors with a low growth fraction. Conclusion: The unique attributes distinguish this approach from other GDEPT strategies and are precisely those required to mediate significant improvements in antitumor therapy.