Alkylglycerol Prodrugs of Phosphonoformate Are Potent In Vitro Inhibitors of Nucleoside-Resistant Human Immunodeficiency Virus Type 1 and Select for Resistance Mutations That Suppress Zidovudine Resistance

Hammond, Jennifer; Koontz, Dianna; Bazmi, Holly; Beadle, James R.; Hostetler, Saskia E; Kini, Ganesh D.; Aldern, Kathy A.; Richman, Douglas D.; Hostetler, Karl Y.; Mellors, John W.

doi:10.1128/aac.45.6.1621-1628.2001

Cited by 42 publications

(30 citation statements)

References 31 publications

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“…This discrepancy is probably due to the limited ability of foscarnet to penetrate into cells, so that high concentrations of extracellular foscarnet are needed to reach inhibitory levels intracellularly. The permeability barrier can be circumvented by the use of alkylglycerol-conjugated derivatives of foscarnet, which are 10-to 35-fold more potent inhibitors of HIV-1 replication than is free foscarnet because they readily penetrate into cells and are then cleaved to form free intracellular foscarnet (13)(14)(15)21). These findings probably explain the differences in foscarnet sensitivity between our cell-based and enzyme assays.…”

Section: Resultsmentioning

confidence: 46%

“…RTs containing both foscarnet and AZT resistance mutations had levels of primer-unblocking activity resulting from the combination of these two opposing effects. This explains why foscarnet-resistant mutants of HIV-1 are often hypersensitive to AZT (31,52) and why AZT resistance mutations are suppressed by foscarnet resistance mutations and visa versa (2,13,54). In vitro resistance to foscarnet by WT and mutant RTs, as measured by effects on [ 3 H]dTMP incorporation, did not match the in vivo resistance measured in cell culture assays ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…Alkyglycerol conjugates of foscarnet have been developed to improve cellular uptake and reduce toxicity (14,15,21). Serial passage of HIV-1 in the presence of these conjugated forms of foscarnet yielded additional mutations including S117T, F160Y, and M164I (usually in combination with the L214F mutation) (13), which also conferred resistance to unconjugated foscarnet in virus culture assays. Finally, the K65R mutation was originally isolated from patients treated with 3Ј-dideoxycytidine or 3Ј-dideoxyinosine (12,59) and subsequently shown to confer resistance to other chain terminators (45) and also to foscarnet (13,48).…”

mentioning

confidence: 99%

“…The mutations W88S, W88G, Q161L, and H208Y were originally identified in clinical isolates of HIV-1 from foscarnet-treated patients (31). W88G, E89K, L92I, S156A, and Q161L/H208Y were obtained by serial passage of HIV in MT-2 cells in the presence of escalating concentrations of foscarnet (13,31,52). Alkyglycerol conjugates of foscarnet have been developed to improve cellular uptake and reduce toxicity (14,15,21).…”

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confidence: 99%

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Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Meyer

Matsuura

Zonarich

et al. 2003

J Virol

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Phosphonoformate (foscarnet) is a pyrophosphate (PP i ) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PP i binding site on the enzyme. HIV-1 RT can unblock a chain-terminated DNA primer by phosphorolytic transfer of the terminal residue to an acceptor substrate (PP i or a nucleotide such as ATP) which also interacts with the PP i binding site. Primer-unblocking activity is increased in mutants of HIV-1 that are resistant to the chain-terminating nucleoside inhibitor 3-azido-3-deoxythymidine (AZT). We have compared the primer-unblocking activity for HIV-1 RT containing various foscarnet resistance mutations (K65R, W88G, W88S, E89K, S117T, Q161L, M164I, and the double mutant Q161L/H208Y) alone or in combination with AZT resistance mutations. The level of primer-unblocking activity varied over a 150-fold range for these enzymes and was inversely correlated with foscarnet resistance and directly correlated with AZT resistance. Based on published crystal structures of HIV-1 RT, many of the foscarnet resistance mutations affect residues that do not make direct contact with the catalytic residues of RT, the incoming deoxynucleoside triphosphate (dNTP), or the primer-template. These mutations may confer foscarnet resistance and reduce primer unblocking by indirectly decreasing the binding and retention of foscarnet, PP i , and ATP. Alternatively, the binding position or orientation of PP i , ATP, or the primer-template may be changed in the mutant enzyme complex so that molecular interactions required for the unblocking reaction are impaired while dNTP binding and incorporation are not.Phosphonoformate (foscarnet) inhibits a wide variety of DNA and RNA polymerases including human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (40, 43). Foscarnet competes with pyrophosphate (PP i ) for binding in PP i exchange reactions, suggesting that foscarnet interacts with the site on the enzyme where PP i is formed (7, 38). On structural grounds, the site of interaction for PP i (and, presumably, for foscarnet) should lie within the deoxynucleoside triphosphate (dNTP) binding site since PP i is formed from the ␤ and ␥ phosphates of the dNTP substrate, but foscarnet inhibition of DNA synthesis in vitro is noncompetitive with dNTP substrates (58), indicating that foscarnet and dNTP bind to distinguishable forms of RT. In addition, median-effect analysis comparing foscarnet and 3Ј-azido-3Ј-deoxythymidine (AZT) triphosphate (AZTTP) alone or as mixtures has shown that inhibition by these compounds is mutually exclusive (10, 22, 50), implying that binding of either inhibitor prevents binding of the other to the same enzyme molecule.In intact cells, the inhibition of HIV-1 by foscarnet and AZT is synergistic (10,22). The mechanism of this synergy is unclear, but it has recently been demonstrated that AZT monophosphate (AZTMP) incorporation can be reversed by the primer-unblocking activity of RT, in which the chain-terminating residue is trans...

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Section: Resultsmentioning

confidence: 46%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Meyer

Matsuura

Zonarich

et al. 2003

J Virol

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“…With few exceptions, mutations that confer decreased susceptibility to NRTIs and NNRTIs do not affect susceptibility to PFA (7)(8)(9). Most importantly, mutations associated with PFA resistance were shown to increase susceptibility to the nucleoside analogue 3Ј-azido-3Ј-deoxythymidine (zidovudine or AZT) (10,11). Biochemical data have shown that PFA-associated resistance mutations diminish the phosphorolytic excision of incorporated chain terminators such as AZT-monophosphate (8,12).…”

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confidence: 99%

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

Marchand

Tchesnokov

Götte

2007

Journal of Biological Chemistry

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The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified "hot spots" for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre-and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. In these cases, the enzyme is seen at multiple positions around the 3 end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation.Different classes of inhibitors that target the reverse transcriptase (RT) 4 of the human immunodeficiency virus type 1 (HIV-1) have been developed (1). Nucleoside analogue reverse transcriptase inhibitors (NRTIs) are major components in drug regimens that are currently used in the clinic. Two different NRTIs are usually combined with a non-nucleoside analogue RT inhibitor (NNRTI) or a protease inhibitor. The triphosphate forms of NRTIs compete with natural nucleotide pools for incorporation, and, once incorporated, the monophosphate acts as a chain terminator. NNRTIs bind to a hydrophobic pocket in the vicinity but not at the active site of HIV-1 RT. Previous studies have suggested that these compounds interfere with the chemical step, whereas nucleotide binding does not appear to be largely affected (2, 3). Here we studied the mechanism of action of the pyrophosphate (PP i ) analogue phosphonoformic acid (PFA or foscarnet), which represents a third class of RT inhibitors.PFA shows a broad spectrum of antiviral activities against various members of the Herpesviridae and Retroviridae (4). The inhibitor is used in the clinic to treat infection with herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), the human cytomegalovirus, and other related herpesviruses when firstline agents have failed (5). Toxic side effects and its poor bioavailability limit its clini...

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Anti‐DNAVirus Agents

Holý

Clercq

2010

Burger's Medicinal Chemistry and Drug Discovery

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Anti‐DNA virus agents that are inhibitory to the replication of DNA viruses have been reviewed by Tim Middleton and Rockway in Burger's Medicinal Chemistry, 6th ed., volume 5. Here we will address the various agents effective against these different viruses. For each of the (classes of) compound(s), we will, where applicable, specifically focus on (i) the antiviral compounds that are clinically available; (ii) the antiviral compounds that are under (pre)clinical development; (iii) the mechanism of action of the compounds; (iv) their structure–activity relationship (SAR); (v) resistance that may have arisen; (vi) recent clinical data obtained with the compounds while under development. Previous reviews on antiviral agents have been dealing with “looking back in 2009 at the dawning of antiviral therapy now 50 years ago: an historical perspective,” highlighting the discovery of acyclovir [9‐(2‐hydroxyethoxymethyl)guanine] as a specific antiherpetic agent, “the design of drugs for HIV and HCV,” “HIV drug development: the next 25 years,” “interferons at age 50: past, current, and future impact on biomedicine,” “the way forward in HCV treatment‐finding the right path,” “antiviral treatment of chronic hepatitis B virus infections: the past, the present, and the future,” “antiviral agents active against influenza A viruses,” “the war against influenza: discovery and development of sialidase inhibitors,” “antivirals and antiviral strategies,” and “clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of DNA virus and retrovirus infections.”

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Alkylglycerol Prodrugs of Phosphonoformate Are Potent In Vitro Inhibitors of Nucleoside-Resistant Human Immunodeficiency Virus Type 1 and Select for Resistance Mutations That Suppress Zidovudine Resistance

Cited by 42 publications

References 31 publications

Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

Anti‐DNAVirus Agents

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