Abstract
Moloney murine leukaemia virus (M-MuLV) is a member of the retrovirus family. Its cloned reverse transcriptase (RT), similarly to HIV type 1 reverse transcriptase (HIV-1 RT), exhibits DNA-polymerase and ribonuclease H (RNase H) activities capable of converting the single-stranded retroviral RNA genome into double-stranded DNA. The latter is then integrated into the host chromosome during viral infection. M-MuLV RT is, therefore, an attractive enzyme to help understand mutations in HIV-1 RT and its use in inhibition studies can help facilitate new drug designs. In this study, conjugates consisting of N-trityl derivatives of p-fluoro, p-nitro and p-iodo-DL-phenylalanine were coupled to 8-(6-aminohexyl) amino-adenosine-3',5'-cyclic monophosphate and examined for their effect on DNA synthesis by M-MuLV RT. Synthesis was studied in a system containing poly (rA).oligo d(pT)15 as a template-primer with [3H] dTTP. The iodo-derivative, N-trityl-p-iodo-DL-phenylalanine-8-(6-aminohexyl) amino-adenosine-3',5'-cyclic monophosphate was found to be a very active inhibitor of the RT enzyme (IC50 = 1 µM), while the p-nitro (IC50 = 45 µM) and p-fluoro (IC50 = 65 µM) were weak inhibitors. Further work will be aimed at determining the mode of binding of the N-tritylated conjugates and also of various substituted amino acids and short peptides to M-MuLV RT to elucidate the mechanisms of inhibition.
Highlights
In a recent article[1] we described the synthesis of a number of N-acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5’-monophosphate
Moloney murine leukaemia virus (M-MuLV) RT is a cloned enzyme expressed in Escherichia coli as a single polypeptide (71 kDa) 2 and, to HIV type 1 reverse transcriptase (HIV-1 RT), exhibits both DNA-polymerase and ribonuclease H (RNase H) activities[3]; the common properties of M-MuLV RT and HIV-1 RT suggest that the two enzymes are able to carry out identical biological functions as far as the mechanism of DNA synthesis is concerned
This paper outlines the synthesis of three compounds containing 8-(6-aminohexyl) amino-adenosine3’,5’-cyclic monophosphate that are linked to different N-tritylp-substituted-DL-phenylalanines (p-fluoro, p-nitro and p-iodo; Figure 2) and their effects on the DNA polymerase activity of M-MuLV RT
Summary
In a recent article[1] we described the synthesis of a number of N-acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5’-monophosphate. N-trityl-m-fluoro-DL-phenylalanine, was attached to the nucleotide 8-(6-aminohexyl) amino-adenosine-5’-monophosphate (Figure 1), the inhibitory activity increased, giving an IC50 value of 5 μM. It was of particular interest to see if replacement of the nucleotide adenosine-5’-phosphate component of N-trityl-p-substituted-DL-phenylalanine-8-(6-aminohexyl) amino-adenosine-5’-monophosphate by adenosine-3’,5’-cyclic monophosphate would result in inhibitory derivatives against the reverse transcriptase.
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