Abstract
Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.
Highlights
Two enantiomers of carbovir, a carbocyclic analog tion of viral DNA synthesis
Stereoselectivity between enanof 2’,3’-dideoxyguanosine, were compared with re- tiomers of carbocyclic nucleosides could be conferred by any spect to their phosphorylation and the phosphorylaotifotnhe phosphorylation steps or by the interaction of triphosof their nucleotides by mammalian enzyme5s”.Nucle- phate with polymerases
To determine why only (-)-carbovir but not (+)-carbovir had anti-HIVactivity, we investigated the enantiomeric specificity of a series of enzymatic phosphorylations of carbovir both enantiomers of carbovir diphosphate at similar [8] (Fig. 1).Two of the phosphorylations involved in the rates
Summary
The enantiomers of carbovir were resolved by using adenosine deaminase-catalyzed deamination of the racemic 2,6diaminopurine analog of carbovir. The inhibition of HIV replication in MT4 cells by carbovir was highly enantioselective. (-)-Carbovir was a potent inhibitor of HIV with an ICs0 of 4.6 p~ f 1.4 p M ( n= 21). Soluble 5"nucleotidase from human placentacatalyzed the phosphorylation of (-)-carbovir. The K , value for (-)-carbovir, 6.1 mM, was similar to thatreported for the 5"nucleotidase from human leukemia cells [20]. (-)-Carbovir was a good substrate with a V,.,/K, value one-third that of inosine (Table I).The enzyme showed a high degree of stereoselectivity for the (-)-enantiomer. Neither enantiomerof carbovir was phosphorylated by deoxycytidine kinase from calf thymus.'. (-)-Carbovir monophosphate was asubstrate for GMP kinase from pig brain (Table 11). The low rate of phosphorylation of (+)-carbovir monophosphate was not due to contamination by the (-)-enantiomer, because formation
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