The Acyclic 2,4-Diaminopyrimidine Nucleoside Phosphonate Acts as a Purine Mimetic in HIV-1 Reverse Transcriptase DNA Polymerization

Brian D Herman,Ivan Votruba,Antonin Holý,Nicolas Sluis-Cremer,Jan Balzarini

doi:10.1074/jbc.m109.096529

Abstract

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.

Highlights

Phonylmethoxyethyl (PME) derivatives (e.g. PMEA); (ii) the phosphonylmethoxypropyl (PMP) derivatives (e.g. (R)PMPA and (S)FPMPA); and (iii) the hydroxyphosphonylmethoxypropyl (HPMP) derivatives (e.g. (S)HPMPA and (S)HPMPC) [1,2,3,4,5]
Acyclic Diaminopyrimidine Nucleoside Phosphonate PMEO-DAPym was supported by molecular modeling and superimposition of their optimized three-dimensional structures, which demonstrated that the 2,4-diamino-substituted pyrimidine could be regarded as an open-ring analog of the purine system in the 2,6-diaminopurine acyclic pyrimidine nucleoside phosphonate (ANP) (Fig. 2) [16]
Consistent with the studies described above, we found that HIV-1 reverse transcriptase (RT) recognized PMEO-DAPym-pp as a purine but not a pyrimidine nucleotide analog lar inhibitory activity (IC50 ϭ 0.26 ␮M), that was similar to PMEA diphosphate (PMEA-pp) (IC50 ϭ 0.12 ␮M) or ddATP (IC50 ϭ 0.23 ␮M), when poly(rU)/oligo(dA) and MnCl2-dATP were used as substrates

Summary

Introduction

Phonylmethoxyethyl (PME) derivatives (e.g. PMEA (adefovir)); (ii) the phosphonylmethoxypropyl (PMP) derivatives (e.g. (R)PMPA (tenofovir) and (S)FPMPA); and (iii) the hydroxyphosphonylmethoxypropyl (HPMP) derivatives (e.g. (S)HPMPA and (S)HPMPC (cidofovir)) [1,2,3,4,5]. The ANP-diphosphate binds to the active site of the viral polymerase and competes with the corresponding natural nucleotide (e.g. dATP for (S)HPMPA, PMEA, (S)FPMPA and (R)PMPA, and dCTP for (S)HPMPC) for subsequent incorporation into the growing DNA chain.

Results

Conclusion