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Synthesis and substrate properties towards HIV-1 reverse transcriptase of new diphosphate analogues of 9-[(2-phosphonomethoxy)ethyl]adenine.

ABSTRACT: Background The replacement of ?,?-pyrophosphate by ?,?-phosphonate moieties within the triphosphate chain of 5'-triphosphate nucleoside analogues was previously studied for various antiviral nucleoside analogues such as AZT and 2',3'-dideoxynucleosides. Thus, it has been shown that these chemical modifications could preserve, in some cases, the terminating substrate properties of the triphosphate analogue for HIV-RT. Herein, we aimed to study such 5'-triphosphate mimics based on the scaffold of the well-known antiviral agent 9-[(2-phosphonomethoxy)ethyl]adenine (PMEA, Adefovir). Methods Synthesis involved coupling of a morpholidate derivative of PMEA with appropriate pyrophosphoryl analogues. The relative efficiencies of incorporation of the studied diphosphate phosphonates were measured using subtype B WT HIV-1 RT in an in vitro susceptibility assay, in comparison to the parent nucleotide analogue (PMEApp). Results Searching for nucleoside 5'-triphosphate mimics, we have synthesized and studied a series of diphosphate analogues of PMEA bearing non hydrolysable bonds between the and phosphorus atoms. We also examined their relative inhibitory capacity towards HIV-1 reverse transcriptase in comparison to the parent nucleotide analogue (PMEApp). Only one of them appeared as a weak inhibitor (IC50?=?403.0?±?75.5?µM) and proved to be less effective than PMEApp (IC50?=?6.4?±?0.8?µM). Conclusion PMEA diphosphoryl derivatives were designed as potential substrates and/or inhibitors of various viral polymerases. These modifications dramatically affect their ability to inhibit HIV-RT.

SUBMITTER: Laux WH

PROVIDER: S-EPMC5890543 | biostudies-literature | 2018 Jan-Dec

REPOSITORIES: biostudies-literature

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Synthesis and substrate properties towards HIV-1 reverse transcriptase of new diphosphate analogues of 9-[(2-phosphonomethoxy)ethyl]adenine.

Laux Wolfgang Hg WH Priet Stéphane S Alvarez Karine K Peyrottes Suzanne S Périgaud Christian C

Antiviral chemistry & chemotherapy 20180101

Background The replacement of β,γ-pyrophosphate by β,γ-phosphonate moieties within the triphosphate chain of 5'-triphosphate nucleoside analogues was previously studied for various antiviral nucleoside analogues such as AZT and 2',3'-dideoxynucleosides. Thus, it has been shown that these chemical modifications could preserve, in some cases, the terminating substrate properties of the triphosphate analogue for HIV-RT. Herein, we aimed to study such 5'-triphosphate mimics based on the scaffold of ...[more]

PMID: 29436843

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Project description:BackgroundDeoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA synthesis catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA synthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics for treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the gamma-phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNDPs in addition to dNTPs.Methodology/principal findingsWe examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K65 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3'-terminal nucleoside inhibitors such as AZT forms the basis for HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3'-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision.ConclusionsWe have identified two new catalytic functions of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3'-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase, the RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated.

Project description:Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.

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Synthesis and substrate properties towards HIV-1 reverse transcriptase of new diphosphate analogues of 9-[(2-phosphonomethoxy)ethyl]adenine.

Publications

Synthesis and substrate properties towards HIV-1 reverse transcriptase of new diphosphate analogues of 9-[(2-phosphonomethoxy)ethyl]adenine.

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