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Therapeutic nucleoside and nucleotide analogs

Since the development and introduction of solid phase oligonucleotide synthesis in the 1950s several nucleoside and nucleotide analogs have been identified as potent antiviral drugs. These compounds exhibit activities against poxviruses including variola, vaccinia, monkeypox, cowpox, molluscum contagiosum, and the orf virus. Nucleoside and nucleotide analogs including fluorescently labled nucleotides can be incorporated synthetically into oligonucleotides using modified phosphoramidites. Depending on the application, short and long modifed oligos can be synthesized using RNA and/or DNA synthesis.

Biosynthesis Inc. offers automated custom synthesis for un-modified and modified short, median length, and long DNA and RNA oligos.

Molecular levels of the nucleoside system including 5’-nucleotidases (5’-NTs) and other nucleoside metabolic enzymes, as well as nucleoside transporters, and receptors, were found to be unevenly distributed in the brain. The nucleosides adenosine (Ado, A), guanosine (Guo, G), inosine (Ino, I) and uridine (Urd, U), are known to modulate both physiological and pathophysiological processes in the brain. We know now that these molecules are important players in the regulation of sleep, pain, memory, depression, schizophrenia, epilepsy, Huntington’s disease, Alzheimer’s disease and Parkinson’s disease.

5,6-dihydro-5-azacytosidine (DZCyt, or DHAC)

Figure 1:  Structures of 5,6-dihydro-5-azacytidine (DHAC) and its phosphoramidite.

Sheikhnejad in 1999 showed how 5,6-dihydro-5-azacytosidine (DZCyt or DHAC), a cytosine analog with a sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, can mimic the non-aromatic character of the cytosine ring in the transition state. The substitution of DZCyt or DHAC for target cytosines in C-G dinucleotides of single-stranded or double-stranded oligodeoxy-ribonucleotide substrates led to complete inhibition of methylation by the murine DNA (cytosine C5)-methyl-transferase (DNA C5-MTase). Substitution of DHAC for the target cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had a similar effect on the methylation by HhaI methyltransferase (M. HhaI).

Oligodeoxyribonucleotides containing DZCyt can form a tight but reversible complex with the enzyme M. HhaI. The compounds are known to be potent inhibitors of DNA methylation. Methyltransferase inhibitors can restore transcriptionally silenced genes and are therefore important molecular tools for current therapies of myelodysplastic syndromes and certain types of leukemias.

A series of 5-azacytidine nucleosides have been investigated recently for their hypomethylation potential (Matoušová et al., 2011). Among these are nucleoside analogs including 5-azacytidine (AC), 2′-deoxy-5-azacytidine (DAC), its α-anomer (α-DAC), 5,6-dihydro-5-azacytidine (DHAC), 2′-deoxy-5,6-dihydro-5-azacytidine (DHDAC, KP-1212), its α-anomer (α-DHDAC), and a 2-pyrimidone ribonucleoside called zebularine. These compounds are now well established, preclinically tested inhibitors of DNA methylation. Protected phosphoramidite monomers are useful for the synthetic incorporation of these monomers into oligonucleotides of various lengths.


Zebularine (1-beta-D-ribofuranosyl-2(1H)-pyrimidinone)

 

Figure 1: Structures of Zebularine, 5-Me-Zebularine, and the phosphoramidite of 5-Me-Zebularine.

Zebularine (1-beta-D-ribofuranosyl-2(1H)-pyrimidinone) is another chemically stable, cytidine analog that displays anti-tumor properties. The molecule acts as a transition-state analog inhibitor of cytidine deaminase by binding to the active size as covalent hydrates. It was also shown to inhibit DNA methylation and tumor growth, both in vitro and in vivo.

The 5-methyl-pyrimidin-2-one, 2’-deoxynucleoside analog has been used as a molecular probe to study the initiation of cellular DNA repair. Zebularine-modified DNA cannot be methylated by the cytosine-[C5]-specific DNA methyltransferases (C5 MTases) M. MSP I and M. Hha I. The m5C-DNA methyltransferases catalyze the transfer of a methyl group from S-adenosyl-L-methionine to the C-5 position of cytosine within the recognition sequence of the substrate DNA. Hhal Methyltransferase modifies the internal cytosine residue (C5) of the sequence GCGC (GC{Me}GC).


The 5-Me-2'-deoxyZebularine-CE Phosphoramidite can be used for the synthesis of oligonucleotides containing this modification. Cytosine DNA methyltransferase inhibitors such as this molecule are known to restore androgen responsiveness in androgen-refractory tumor cells. The treated tumor cells are then sensitive to growth inhibition by anti-androgens.

Cytosine arabinoside (araC)


 

Figure 3: Structures for araC and its phosphoramidite.

Cytosine arabinoside (araC), a pyrimidine analogue, is one of the most effective agents or drugs used in the treatment of acute leukemia. araC is thought to exert its chemotherapeutic activity by the inhibition of the nuclear factor NF-κB. However, araC needs to be incorporated into chromosomal DNA to have a cytotoxic effect. The drug is converted to its “active” nucleoside triphosphate form by the pyrimidine salvage pathways. After integration into chromosomes, the arabinoside inhibits chain elongation and bypasses synthesis. In some cases, araC can induce DNA chain termination or duplication of DNA sequences.

However, the precise mechanism of araC-induced cell death may still need to be established. Apparently, treatment with this drug leads to reduced rates of DNA replication, DNA strand breaks, and chromosome fragmentation.

Reference


Susan D. Cline and Neil Osheroff; Cytosine Arabinoside Lesions Are Position-specific Topoisomerase II Poisons and Stimulate DNA Cleavage Mediated by the Human Type II Enzymes. Vol. 274, No. 42, Issue of October 15, pp. 29740 –29743, 1999.

De Clercq E, Neyts J.; Therapeutic potential of nucleoside/nucleotide analogues against poxvirus infections. Rev Med Virol. 2004 Sep-Oct;14(5):289-300.


Drugbank: 
http://www.drugbank.ca/drugs/DB03068

Kovács Z, Dobolyi A, Kékesi KA, Juhász G.; 5'-nucleotidases, nucleosides and their distribution in the brain: pathological and therapeutic implications.
Curr Med Chem. 2013;20(34):4217-40.

Kumar S, Cheng X, Klimasauskas S, Mi S, Posfai J, Roberts RJ, Wilson GG.; The DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 1994 Jan 11;22(1):1-10.

Matoušová M, Votruba I, Otmar M, Tloušťová E, Günterová J, Mertlíková-Kaiserová H. 2′-deoxy-5,6-dihydro-5-azacytidine—a less toxic alternative of 2′-deoxy-5-azacytidine: A comparative study of hypomethylating potential. Epigenetics. 2011;6(6):769-776. doi:10.4161/epi.6.6.16215.

David P. Martin, Thomas L. Wallace, and Eugene M. Johnson, Jr.; Cytosine Arabinoside Kills Postmitotic Neurons in a Fashion Resembling Trophic Factor Deprivation: Evidence That a Deoxycytidine-Dependent Process May Be Required forNerve Growth Factor Signal Transduction. The Journal of Neuroscience, January 1990, IO(l): 184-193.

Gholamreza Sheikhnejad, Adam Brank, Judith K. Christman, Amanda Goddard, Estela Alvarez, Harry Ford Jr, Victor E. Marquez, Canio J. Marasco, Janice R. Sufrin, Margaret O'Gara and Xiaodong Cheng; Mechanism of Inhibition of DNA (Cytosine C5)-Methyltransferases by  Oligodeoxyribo-nucleotides Containing 5,6-Dihydro-5-azacytosine. J. Mol. Biol. (1999) 285, 2021-2034.

Singleton SF, Roca AI, Lee AM, Xiao J. Probing the structure of RecA-DNA filaments. Advantages of a fluorescent guanine analog. Tetrahedron. 2007;63(17):3553-3566.  doi:10.1016/j.tet.2006.10.092.

Zhou L, Cheng X, Connolly BA, Dickman MJ, Hurd PJ, Hornby DP. Zebularine: A Novel DNA Methylation Inhibitor that Forms a Covalent Complex with DNA Methyltransferases. Journal of molecular biology. 2002;321(4):591-599. doi:10.1016/S0022-2836(02)00676-9.

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