ABSTRACT
Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.
Subject(s)
Drug Resistance, Viral/physiology , HIV Reverse Transcriptase/chemistry , HIV-1/drug effects , Reverse Transcriptase Inhibitors/pharmacology , Zidovudine/pharmacology , Adenosine Triphosphate/metabolism , Amino Acid Substitution , Binding Sites/drug effects , Crystallography, X-Ray , DNA, Viral/biosynthesis , Deoxyribonucleotides/metabolism , Dideoxynucleotides/metabolism , Drug Design , Drug Resistance, Viral/genetics , Genes, rev , HIV Reverse Transcriptase/genetics , HIV-1/enzymology , HIV-1/genetics , Models, Molecular , Mutation, Missense , Point Mutation , Protein Conformation , Structure-Activity Relationship , Thymine Nucleotides/metabolism , Zidovudine/analogs & derivatives , Zidovudine/metabolismABSTRACT
We report efficient, one-flask procedures for the synthesis of a family of fourteen analogs of AZTp(4)A and Ap(4)A containing BH(3), S, or Se, along with two bisphosphonate analogs of Ap(4)A. These compounds should slow unwanted enzymatic hydrolysis and have the potential to create unique binding interactions in biochemical and structural studies of the excision reaction responsible for resistance of HIV-1 to AZT, as well as assist in drug design.
ABSTRACT
(15)N NMR chemical shift changes in the presence of Mg(H(2)O)(6)(2+), Zn(2+), Cd(2+), and Co(NH(3))(6)(3+) were used to probe the effect of flanking bases on metal binding sites in three different RNA motifs. We found that: for GC pairs, the presence of a flanking purine creates a site for the soft metals Zn(2+) and Cd(2+) only; a GG.UU motif selectively binds only Co(NH(3))(6)(3+), while a UG.GU motif binds none of these metals; a 3' guanosine flanking the adenosine of a sheared GA.AG pair creates an unusually strong binding site that precludes binding to the cross-strand stacked guanosines within the tandem pair.
Subject(s)
Base Pairing , Metals/metabolism , RNA/chemistry , RNA/metabolism , Binding Sites , Nuclear Magnetic Resonance, Biomolecular , RNA/chemical synthesisABSTRACT
We report an efficient, one-flask route for synthesis of AZTpSpCX2ppSA and AZTpSpCX2ppSAZT, where X=H and X=F. This route makes use of the differential susceptibility to oxidation of H-phosphonate mono- and diesters, to allow a series of sequential reactions without requiring isolation of intermediates. These compounds are hydrolysis-resistant versions of the AZTppppA that results from excision of AZT by AZT-resistant HIV reverse transcriptase (RT). This family of compounds may therefore be useful in further study of the AZT excision reaction, as well as in drug design.
Subject(s)
HIV Reverse Transcriptase/antagonists & inhibitors , Reverse Transcriptase Inhibitors/chemical synthesis , Zidovudine/analogs & derivatives , Zidovudine/chemical synthesis , HIV Reverse Transcriptase/metabolism , Hydrolysis , Molecular Structure , Phosphates/chemistry , Reverse Transcriptase Inhibitors/chemistry , Zidovudine/chemistryABSTRACT
[reaction: see text] We report a one-flask route for the synthesis of dinucleoside tetra- and pentaphosphates, in isolated yields of 50-85%. This route relies on a mixture of P(III) and P(V) chemistries, using phosphitylation of a protected nucleoside with 2-chloro-4H-l,3,2-benzo-dioxaphosphorin-4-one (salicylchlorophosphite), followed by sequential reaction with inorganic pyrophosphate and a nucleoside 5' mono- or diphosphate.