scholarly journals The Phenylmethylthiazolylthiourea Nonnucleoside Reverse Transcriptase (RT) Inhibitor MSK-076 Selects for a Resistance Mutation in the Active Site of Human Immunodeficiency Virus Type 2 RT

2004 ◽  
Vol 78 (14) ◽  
pp. 7427-7437 ◽  
Author(s):  
Joeri Auwerx ◽  
Miguel Stevens ◽  
An R. Van Rompay ◽  
Louise E. Bird ◽  
Jingshan Ren ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Resistance Mutation ◽  
Site Directed Mutagenesis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 μM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 μM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC)·oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.

scholarly journals Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA

2004 ◽  
Vol 78 (7) ◽  
pp. 3387-3397 ◽  
Author(s):  
Elena N. Peletskaya ◽  
Alex A. Kogon ◽  
Steven Tuske ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Cross Linking ◽  
Immunodeficiency Virus ◽  
Dntp Binding ◽  
Hiv 1

ABSTRACT Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the “open” configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

scholarly journals Inhibition of Foamy Virus Reverse Transcriptase by Human Immunodeficiency Virus Type 1 RNase H Inhibitors

2014 ◽  
Vol 58 (7) ◽  
pp. 4086-4093 ◽  
Author(s):  
Angela Corona ◽  
Anna Schneider ◽  
Kristian Schweimer ◽  
Paul Rösch ◽  
Birgitta M. Wöhrl ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Foamy Virus ◽  
Rnase H ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTRNase H plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1). Therefore, it is a promising target for drug development. However, the identification of HIV-1 RNase H inhibitors (RHIs) has been hampered by the open morphology of its active site, the limited number of available RNase H crystal structures in complex with inhibitors, and the fact that, due to the high concentrations of Mg2+needed for protein stability, HIV-1 RNase H is not suitable for nuclear magnetic resonance (NMR) inhibitor studies. We recently showed that the RNase H domains of HIV-1 and prototype foamy virus (PFV) reverse transcriptases (RTs) exhibit a high degree of structural similarity. Thus, we examined whether PFV RNase H can serve as an HIV-1 RNase H model for inhibitor interaction studies. Five HIV-1 RHIs inhibited PFV RNase H activity at low-micromolar concentrations similar to those of HIV-1 RNase H, suggesting pocket similarity of the RNase H domains. NMR titration experiments with the PFV RNase H domain and the RHI RDS1643 (6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester) were performed to determine its binding site. Based on these results and previous data,in silicodocking analysis showed a putative RDS1643 binding region that reaches into the PFV RNase H active site. Structural overlays were performed with HIV-1 and PFV RNase H to propose the RDS1643 binding site in HIV-1 RNase H. Our results suggest that this approach can be used to establish PFV RNase H as a model system for HIV-1 RNase H in order to identify putative inhibitor binding sites in HIV-1 RNase H.

scholarly journals Genotypic, Phenotypic, and Modeling Studies of a Deletion in the β3-β4 Region of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Gene That Is Associated with Resistance to Nucleoside Reverse Transcriptase Inhibitors

2000 ◽  
Vol 74 (22) ◽  
pp. 10707-10713 ◽  
Author(s):  
Mark A. Winters ◽  
Kristi L. Coolley ◽  
Peng Cheng ◽  
Yvette A. Girard ◽  
Hasnah Hamdan ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Point Mutations ◽  
Nucleoside Analogs ◽  
Site Directed Mutagenesis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Point mutations and inserts in the β3-β4 region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are associated with resistance to nucleoside analog inhibitors. This report describes HIV-1 strains from seven patients that were found to have a 3-bp deletion in the β3-β4 region of the RT gene. These patient strains also had a mean of 6.2 drug resistance-associated mutations in their RT genes (range, 3 to 10 mutations). The deletion was most frequently found in strains with the Q151M mutation. Nonnucleoside RT inhibitor mutations were found in six of seven strains. Culture-based drug sensitivity assays showed that deletion-containing isolates had reduced susceptibility to four to eight RT inhibitors. Site-directed mutagenesis experiments showed that the deletion alone conferred reduced susceptibility to nucleoside analogs. Changes in the three-dimensional models of the RT deletion mutants were consistently observed at the β3-β4 loop and at helices C and E in both the presence and the absence of dTTP. Loss of hydrogen bonds between the RT and dTTP were also observed in the RT deletion mutant. These results suggest that the deletion in the RT gene contributes to resistance to several nucleoside analogs through a complex interaction with other mutations in the RT gene.

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

2003 ◽  
Vol 77 (11) ◽  
pp. 6127-6137 ◽  
Author(s):  
Peter R. Meyer ◽  
Suzanne E. Matsuura ◽  
Dianna Zonarich ◽  
Rahul R. Chopra ◽  
Eric Pendarvis ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Phosphonoformate (foscarnet) is a pyrophosphate (PPi) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PPi 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 (PPi or a nucleotide such as ATP) which also interacts with the PPi 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, PPi, and ATP. Alternatively, the binding position or orientation of PPi, 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.

scholarly journals A Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutational Pattern Confers Phenotypic Lamivudine Resistance in the Absence of Mutation 184V

2000 ◽  
Vol 44 (3) ◽  
pp. 568-573 ◽  
Author(s):  
Kurt Hertogs ◽  
Stuart Bloor ◽  
Veronique De Vroey ◽  
Christel van den Eynde ◽  
Pascale Dehertogh ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Site Directed Mutagenesis ◽  
Phenotypic Resistance ◽  
Mutational Pattern ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4- and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.

scholarly journals Cross-Linking of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Template-Primer

2001 ◽  
Vol 75 (19) ◽  
pp. 9435-9445 ◽  
Author(s):  
Elena N. Peletskaya ◽  
Paul L. Boyer ◽  
Alex A. Kogon ◽  
Patrick Clark ◽  
Heiko Kroth ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Cross Linking ◽  
Cysteine Residues ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Cross-linking experiments were performed with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Two approaches were used—photoaffinity cross-linking and disulfide chemical cross-linking (using an oligonucleotide that contained an N2-modified dG with a reactive thiol group). In the former case, cross-linking can occur to any nucleotide in either DNA strand, and in the latter case, a specific cross-link is produced between the template and the enzyme. Neither the introduction of the unique cysteine residues into the fingers nor the modification of these residues with photocross-linking reagents caused a significant decrease in the enzymatic activities of RT. We were able to use this model system to investigate interactions between specific points on the fingers domain of RT and double-stranded DNA (dsDNA). Photoaffinity cross-linking of the template to the modified RTs with Cys residues in positions 65, 67, 70, and 74 of the fingers domain of the p66 subunit was relatively efficient. Azide-modified Cys residues produced 10 to 25% cross-linking, whereas diazirine modified residues produced 5 to 8% cross-linking. Disulfide cross-linking yields were up to 90%. All of the modified RTs preferentially photocross-linked to the 5′ extended template strand of the dsDNA template-primer substrate. The preferred sites of interactions were on the extended template, 5 to 7 bases beyond the polymerase active site. HIV-1 RT is quite flexible. There are conformational changes associated with substrate binding. Cross-linking was used to detect intramolecular movements associated with binding of the incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreases the efficiency of cross-linking, but causes only modest changes in the preferred positions of cross-linking. This suggests that the interactions between the fingers of p66 and the extended template involve the “open” configuration of the enzyme with the fingers away from the active site rather than the closed configuration with the fingers in direct contact with the incoming dNTP. This experimental approach can be used to measure distances between any site on the surface of the protein and an interacting molecule.

scholarly journals Amino Acid Mutation N348I in the Connection Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Multiclass Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors

2008 ◽  
Vol 82 (7) ◽  
pp. 3261-3270 ◽  
Author(s):  
Atsuko Hachiya ◽  
Eiichi N. Kodama ◽  
Stefan G. Sarafianos ◽  
Matthew M. Schuckmann ◽  
Yasuko Sakagami ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resistance Mutation ◽  
Dependent Manner ◽  
Reverse Transcriptase Inhibitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We identified clinical isolates with phenotypic resistance to nevirapine (NVP) in the absence of known nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations. This resistance is caused by N348I, a mutation at the connection subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Virologic analysis showed that N348I conferred multiclass resistance to NNRTIs (NVP and delavirdine) and to nucleoside reverse transcriptase inhibitors (zidovudine [AZT] and didanosine [ddI]). N348I impaired HIV-1 replication in a cell-type-dependent manner. Acquisition of N348I was frequently observed in AZT- and/or ddI-containing therapy (12.5%; n = 48; P < 0.0001) and was accompanied with thymidine analogue-associated mutations, e.g., T215Y (n = 5/6) and the lamivudine resistance mutation M184V (n = 1/6) in a Japanese cohort. Molecular modeling analysis shows that residue 348 is proximal to the NNRTI-binding pocket and to a flexible hinge region at the base of the p66 thumb that may be affected by the N348I mutation. Our results further highlight the role of connection subdomain residues in drug resistance.

scholarly journals Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

2006 ◽  
Vol 50 (8) ◽  
pp. 2772-2781 ◽  
Author(s):  
Zhijun Zhang ◽  
Michelle Walker ◽  
Wen Xu ◽  
Jae Hoon Shim ◽  
Jean-Luc Girardet ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
Nonnucleoside Inhibitors ◽  
Rt Inhibitors ◽  
Hiv 1 ◽  
M184v Mutation

ABSTRACT Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.

scholarly journals The Thiocarboxanilide Nonnucleoside Inhibitor UC781 Restores Antiviral Activity of 3′-Azido-3′-Deoxythymidine (AZT) against AZT-Resistant Human Immunodeficiency Virus Type 1

1999 ◽  
Vol 43 (2) ◽  
pp. 259-263 ◽  
Author(s):  
Gadi Borkow ◽  
Dominique Arion ◽  
Mark A. Wainberg ◽  
Michael A. Parniak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT N-[4-Chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide (UC781) is an exceptionally potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. We found that a 1:1 molar combination of UC781 and 3′-azido-3′-deoxythymidine (AZT) showed high-level synergy in inhibiting the replication of AZT-resistant virus, implying that UC781 can restore antiviral activity to AZT against AZT-resistant HIV-1. Neither the nevirapine plus AZT nor the 2′,5′-bis-O-(t-butyldimethylsilyl)-3′-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide plus AZT combinations had this effect. Studies with purified HIV-1 reverse transcriptase (from a wild type and an AZT-resistant mutant) showed that UC781 was a potent inhibitor of the pyrophosphorolytic cleavage of nucleotides from the 3′ end of the DNA polymerization primer, a process that we have proposed to be critical for the phenotypic expression of AZT resistance. Combinations of UC781 plus AZT did not act in synergy to inhibit the replication of either wild-type virus or UC781-resistant HIV-1. Importantly, the time to the development of viral resistance to combinations of UC781 plus AZT is significantly delayed compared to the time to the development of resistance to either drug alone.

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