A Mechanism of AZT Resistance

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...

Section: Discussionsupporting

confidence: 80%

mentioning

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

“…The other class of resistance mechanism involves repair (or unblocking) of the analog-terminated DNA chain, using PP i or a nucleoside 5Ј-NTP as a cosubstrate for the pyrophosphorolysis reaction (4,7). Mutations in RT such as M41L, D67N, K70R, L210W, T215Y/F, or K219Q/E/N (referred as thymidine analog-associated mutations) increase the unblocking activity in the case of resistance to AZT and d4T (32,33).…”

Section: Discussionmentioning

confidence: 99%

Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using α-Boranophosphate Nucleoside Analogs

Deval

Alvarez

Selmi

et al. 2005

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that ␣-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH 3 -d4TTP and BH 3 -3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3-5-fold resistance to d4TTP. This resistance is suppressed using BH 3 -d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH 3 -3TCTP because of a 160-fold acceleration of the catalytic constant k pol . Mechanistic insights into the rate enhancement were obtained using various ␣-boranophosphate nucleotides. The presence of the BH 3 group renders k pol independent of amino acid substitutions present in RT. Indeed, the ϳ100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH 3 -dTTP. Metal ion titration studies show that ␣-boranophosphate nucleoside analogs enhance 3-8-fold the binding of Mg 2؉ ions to the active site of the RT⅐DNA⅐dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.

“…Excision of AZT-MP occurs with considerable efficiency with wild-type HIV-1 RT, and the rate of pyrophosphorolysis appeared to be increased with resistant RT enzymes (1). The difference in regard to efficiency of the excision reaction between both enzymes is dramatically increased when NTPs act as a pyrophosphate donor (3). In the light of these findings it is now widely accepted that ATP can act as a physiologically relevant pyrophosphate donor.…”

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confidence: 96%

“…Previous biochemical studies have shown that the incorporated chain terminator can be removed from the primer terminus through phosphorolytic cleavage in the presence of either pyrophosphate (PP i ) (1) or in the presence of ribonucleotide triphosphates (NTPs) (2), which were shown to act as pyrophosphate donors. Increasing evidence suggests that the latter reaction pathway provides an important mechanism for HIV resistance to AZT and, to a certain degree, also to other NRTIs (3)(4)(5)(6)(7)(8)(9)(10)(11).…”

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confidence: 99%

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

Marchand

Götte

2003

146

Resistance to nucleoside analogue inhibitors of the reverse transcriptase of the HIV-1 often involves phosphorolytic excision of the incorporated chain terminator. Previous crystallographic and modeling studies suggested that this reaction could only occur when the enzyme resides in a pre-translocational stage. Here we studied mechanisms of polymerase translocation using novel site-specific footprinting techniques. Classical footprinting approaches, based on the detection of protected nucleic acid residues, are not sensitive enough to visualize subtle structural differences at single nucleotide resolution. Thus, we developed chemical footprinting techniques that give rise to hyperreactive cleavage on the template strand mediated through specific contacts with the enzyme. Two specific cuts served as markers that defined the position of the polymerase and RNase H domain, respectively. We show that the presence of the next correct dNTP, following the incorporated chain terminator, caused a shift in the position of the two cuts a single nucleotide further downstream. The footprints point to monotonic sliding motions and provide compelling evidence for the existence of an equilibrium between pre-and post-translocational stages. Our data show that enzyme translocation is reversible and uncoupled from nucleotide incorporation and the release of pyrophosphate. This translocational equilibrium ensures access to the pre-translocational stage after incorporation of the chain terminator. The efficiency of excision correlates with an increase in the population of complexes that exist in the pre-translocational stage, and we show that the latter configuration is preferred with an enzyme that contains mutations associated with resistance to nucleoside analogue inhibitors.