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Novel Approach to Fight Cancer

By: Carol A. Rouzer, VICB Communications
Published: September 23, 2010


Nucleoside analogs with a rigid structure selectively target DNA polymerases that are highly expressed in malignant cells.

DNA encodes the information by which all organisms live and reproduce.  Yet the integrity of DNA is threatened constantly by physical and chemical agents that are present in the environment or produced during normal metabolic processes.  As a result of exposure to these agents, a cell may experience up to one million structure-altering molecular lesions in its DNA per day.  Because it is so important that DNA be replicated flawlessly, the DNA polymerase enzymes that normally carry out this process are stringently designed to recognize only the intact, native DNA structure.  As a result, problems arise in the case of damaged DNA which, if not repaired, may completely block the replication process and lead to cell death.  To avoid this devastating outcome, cells express a special class of translesion polymerases that are capable of replicating DNA even in the presence of damaged bases or unusual secondary structure, but which lack the high fidelity of the primary replicative polymerases.  The Y family is an important group of translesion polymerases as illustrated by the fact that mutation of one family member, DNA polymerase η (hPol η), leads to xeroderma pigmentosum variant type, a disease characterized by a high susceptibility to skin cancer.  In addition, aberrant function of certain Y family polymerases has been associated with cancers of the breast, ovary, colon, and lung.

                

Figure 1. Structures of N-MC-dA and S-MC-dA.


The fact that Y family polymerases may play a role in cancer suggests that small molecule stimulators or inhibitors of their activity may prove to be effective anti-tumor agents. VICB member Martin Egli, in collaboration with Fred Guengerich and Larry Marnett are seeking to exploit that possibility in their exploration of the interaction of nucleoside analogs with three Y family polymerases [Eoff et al. (2010) Angew Chem Int Ed Engl, published online Sept. 2, DOI: 10.1002/anie.201003168]. Nucleoside-based drugs have been employed in the past to inhibit polymerase activity as illustrated by the use of 3′-azido-2′-deoxythymidine (AZT) to block viral replicative polymerases, particularly in the case of HIV infection.  AZT and similar drugs are taken up by the cell and converted into nuceloside triphosphates, which are then incorporated into a nascent DNA chain in place of the corresponding normal nucleotide.  Once incorporated, the drug blocks further replication by preventing the addition of the next nucleotide to the chain.  The resulting failure of DNA replication leads to cell death.


Unfortunately, experience with nucleoside-based agents has shown that drug resistance is readily achieved by mutations in the target polymerase that enable it to excise the drug from the growing DNA chain.  Because excision is made easier by flexibility in the nucleoside molecule, medicinal chemists are designing new nucleoside analogs with rigid structures in order to limit the development of drug resistance.  One way to achieve a rigid structure is to synthesize nucleosides based on a bicyclo[3.1.0]hexane scaffold.  The scaffold replaces the nucleoside’s deoxyribofuranose moiety with a rigid ring locked in a North (N) or South (S) orientation.  The resulting analogs, designated methanocarba-2′-deoxynucleosides (MC-dN, where N represents the attached DNA base) have markedly different three dimensional structures that resemble the conformations of A-form or B-form DNA, respectively.  A notable difference is seen in the position of the 3′ hydroxyl group, which is equatorial in N orientation nucleosides and axial in S orientation nucleosides, as illustrated for adenine-containing analogs in Figure 1 (above).

Figure 2.  Cartoon representation of the structure of the Pol η active site.
Image provided by the European Bioinformatics Institute (http://www.ebi.ac.uk/) and obtained through Wikimedia Commons under the GNU Free Documentation License.


Previous studies have shown that DNA polymerases vary considerably with regard to their ability to incorporate nucleotide triphosphates formed from MC-dNs into DNA.  Enzymes studied thus far strongly favor N-MC-dNTPs over S-MC-dNTPs.  To determine the potential of these molecules to serve as modulators of Y family polymerases, the Egli, Guengerich, and Marnett labs evaluated the effects of the adenine-containing nucleotides (N-MC-dATP and S-MC-dATP) with Y family members hPol η, hPol ι, and hPol κ.  The investigators found that the three enzymes differed substantially in their ability to incorporate the MC-dATP analogs into a synthetic template primer opposite dT, and to extend the chain beyond the added nucleoside.  hPol κ most closely resembled previously studied polymerases in that it could both incorporate and extend beyond N-MC-dATP but not S-MC-dATP.  Incorporation efficiency of a single N-MC-dATP was approximately 25% that of dATP.  In contrast, hPol η could incorporate and extend beyond both MC-dATPs; however, the efficiency of incorporation of a single nucleotide was reduced for both analogs.  hPol η utilized N-MC-dATP and S-MC-dATP with efficiencies that were 25% and 1.2% that of dATP incorporation.  The striking finding with hPol ι was that its efficiency of incorporation of a single N-MC-dATP was 5-fold higher than that of dATP.

Figure 3. Cartoon representation of the structure of the Pol ι active site.
Image provided by the European Bioinformatics Institute (http://www.ebi.ac.uk/) and obtained through Wikimedia Commons under the GNU Free Documentation License.


The remarkable differences in MC-dATP utilization among the three Y family polymerases is not a total surprise.  The investigators note that hPol ι is exceptional in its tendency to incorporate dGTP rather than the expected dATP opposite dT.  They postulate that hPol ι’s ability to utilize N-MC-dATP with such high efficiency may be due to the fact that the preferred conformation for both N-MC-dATP and dGTP is syn as opposed to the anti conformation preferred by dATP.  In the case of, hPol η structural studies have shown that substrate DNA assumes the B-form in the enzyme active site.  This may explain hPol η’s unusual ability to utilize S-MC-dATP, which assumes the conformation of B-form DNA.


The exceptional activity of hPol ι with N-MC-dATP led the team of investigators to explore the ability of this analog to inhibit the growth of tumor cells expressing high levels of hPol ι.  Indeed, the malignant Hs578T breast cancer cell line was sixteen times more sensitive to growth inhibition by N-MC-dATP than the nonmalignant Hs578Bst cell line, a finding that correlated with the observation that Hs578T cells express hPol ι at levels three-fold higher than those of Hs578Bst cells.  These promising results suggest that MC-dN-based drugs could prove to be effective agents against certain cancers with limited toxicity to normal cells, a possibility that is being actively explored by the Egli/Guengerich/Marnett team.

 






                                                          

 

 


                                                            

 



 

 


 

 


 

 
     

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