NUCLEOTIDE AND NUCLEOSIDE ANALOGS AS INHIBITORS OF CYTOSOLIC 5'-NUCLEOTIDASE-I FROM HEART

Substrate and product specificity studies were used to develop inhibit ors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium. As mea sured by V-max/K-m, c-N-I preferred pyrimidine 2'-deoxyribonucleotides as substrates with thymidine monophosphate (TMP) being the most effic ient. In product inhibition studies, thymidine inhibited noncompetitiv ely and inorganic phosphate inhibited competitively, consistent with a n ordered release of nucleoside prior to phosphate. Mirroring nucleoti de substrate specificities, pyrimidine nucleosides were more potent pr oduct inhibitors than purine nucleosides. Thus, pyrimidine nucleotide and nucleoside analogues were developed as inhibitors. Phosphonate ana logues of TMP were synthesized by a novel method. The most potent was the 5'-phosphonate of 3'-deoxythymidine (ddT) (apparent K-i value of 6 3 nM). In addition, pyrimidine nucleoside analogues were inhibitors wi th 5-ethynyl-2',3'-dideoxyuridine being the most potent (apparent K-i value of 3.7 mu M) The most potent nucleotide and nucleoside inhibitor were both greater than 1000-fold more potent inhibiting c-N-I than th e cytosolic 5'-nucleotidase II. The nucleoside analogue was also great er than 1000-fold more potent against c-N-I than the membrane ecto-5'- nucleotidase (e-N). Because the phosphonate analogues measurably inhib ited e-N (apparent K-i values of 6-12 mu M), the selectivity of the ph osphonates for c-N-I versus e-N was less (40-200-fold). Because of the high selectivity for c-N-I versus both of the other 5'-nucleotidases, the nucleoside inhibitors of c-N-I may be useful biochemical tools in discerning the role that c-N-I plays in generating adenosine within m yocardium.