Tenofovir-Amino Acid Conjugates Act as Polymerase SubstratesImplications for Avoiding Cellular Phosphorylation in the Discovery of Nucleotide Analogues

Nucleotide analogues are used for treating viral infections such
as HIV, hepatitis B, hepatitis C, influenza, and SARS-CoV-2. To become
polymerase substrates, a nucleotide analogue must be phosphorylated
by cellular kinases which is rate-limiting. The goal of this study
is to develop dNTP/NTP analogues directly from nucleotides. Tenofovir
(TFV) analogues were synthesized by conjugating with amino acids.
We demonstrate that some conjugates act as dNTP analogues and HIV-1
reverse transcriptase (RT) catalytically incorporates the TFV part
as the chain terminator. X-ray structures in complex with HIV-1 RT/dsDNA
showed binding of the conjugates at the polymerase active site, however,
in different modes in the presence of Mg2+ versus Mn2+ ions. The adaptability of the compounds is seemingly essential
for catalytic incorporation of TFV by RT. 4d with a carboxyl
sidechain demonstrated the highest incorporation. 4e showed
weak incorporation and rather behaved as a dNTP-competitive inhibitor.
This result advocates the feasibility of designing NTP/dNTP analogues
by chemical substitutions to nucleotide analogues.