Carbocyclic Nucleoside Isosteres as Potential Antitumor

Basic Sciences

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Abstract

The main objective of this project has been the systematic probing of nucleoside/nucleotide binding enzymes with sets of conformationally rigid substrates to learn about their preferred binding mode. This has been accomplished by constructing carbocyclic nucleoside analogues on a bicyclo3.1.0hexane template which essentially removes the inherent flexibility of the sugar moiety by locking the conformation of the sugar in one of the two preferred natural conformations (North or South). A secondary objective of this project has been the construction of modified nucleic acids that incorporate a number of locked nucleotide units to either reinforce or disrupt the typical B- or A-DNA conformations associated with South and North conformations, respectively. The major findings are:1) As with the 5'-triphosphate of the locked adenosine analogue (North-MCdA) disclosed last year, the newly synthesized thymine analogue (North-MCT) was also very effective in inhibiting HIV RT beyond the polymerase site and showed great effectiveness against all HIV-resistant strains that function by the excision mechanism. This approach, which offers a novel and promising way to overcome HIV resistance, was successful in treating HIV-infected HOS cells that were transfected with the herpes kinase gene. An effective mean to deliver the monophosphate pro-drug of either the adenine or thymine analogues are being investigated to avoid the use of gene transfection.2) A series of short oligodeoxynucleotides (ODNs) corrresponding to the self-complementary EcoR1 recognition sequence ds(5'-CGCGAATTCGCG-3') where the middle T's were replaced by locked thymidines were synthesized and the structures solved by NMR, CD and calorimetry. NMR studies in a filamentous phage anisotropic medium permitted the measurement of residual dipolar couplings which demonstrated an additive-induced bending of the DNA commensurate with the number of locked thymidine analogues present. 3) The most exciting compound of this series, North-MCT was found to be very active against human herpesvirus 8, a gamma-2 herpesvirus and causative agent of Kaposi's sarcoma, and two in vitro models predictive of activity against small-pox. Intellectual protection on these two findings was initiated this year. 4) Of the two locked analogues of cytosine built on a bicyclo3.1.0hexane template, it was the South analogue the preferred substrate for cytidine deaminase. This result is opposite to the preference shown by the mechanistically similar adenosine deaminase enzyme where the preference is clearly for the North adenine analogue.