Characterization of HIV Reverse Transcriptases with Tyr181→Cys and Leu100→lle Mutations

Zhang, H.; Vrang, Lotta; Unge, Torsten; Öberg, Bo

doi:10.1177/095632029300400506

Cited by 17 publications

(15 citation statements)

References 36 publications

(24 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Crystallization was performed by vapor diffusion as follows. Drops consisting of 5 lL premixed RT (20 Data collection and processing, structure solution and refinement X-ray data were collected at 4°C using the Max-Laboratory beam line 711 and ESRF beam line BM14. Indexing and integration of data were performed using DENZO, the data were merged together with SCALEPACK [21], and further processing was performed with the CCP4 program suite [22].…”

Section: A T E R I a L S A N D M E T H O D Smentioning

confidence: 99%

See 1 more Smart Citation

Structural basis for the inhibitory efficacy of efavirenz (DMP‐266), MSC194 and PNU142721 towards the HIV‐1 RT K103N mutant

Lindberg

Sigurðsson

Löwgren

et al. 2002

European Journal of Biochemistry

135

View full text Add to dashboard Cite

The K103N substitution is a frequently observed HIV-1 RT mutation in patients who do not respond to combinationtherapy. The drugs Efavirenz, MSC194 and PNU142721 belong to the recent generation of NNRTIs characterized by an improved resistance profile to the most common single point mutations within HIV-1 RT, including the K103N mutation. In the present study we present structural observations from Efavirenz in complex with wild-type protein and the K103N mutant and PNU142721 and MSC194 in complex with the K103N mutant. The structures unanimously indicate that the K103N substitution induces only minor positional adjustments of the three inhibitors and the residues lining the binding pocket. Thus, compared to the corresponding wild-type structures, these inhibitors bind to the mutant in a conservative mode rather than through major rearrangements. The structures implicate that the reduced inhibitory efficacy should be attributed to the changes in the chemical environment in the vicinity of the substituted N103 residue. This is supported by changes in hydrophobic and electrostatic interactions to the inhibitors between wild-type and K103N mutant complexes. These potent inhibitors accommodate to the K103N mutation by forming new interactions to the N103 side chain. Our results are consistent with the proposal by Hsiou et al. [Hsiou, Y., Ding, J., Das, K., Clark, A.D. Jr, Boyer, P.L., Lewi, P., Janssen, P.A., Kleim, J.P., Rosner, M., Hughes, S.H. & Arnold, E. (2001) J. Mol. Biol. 309, 437-445] that inhibitors with good activity against the K103N mutant would be expected to have favorable interactions with the mutant asparagines side chain, thereby compensating for resistance caused by stabilization of the mutant enzyme due to a hydrogen-bond network involving the N103 and Y188 side chains.Keywords: drug-resistance; HIV; NNRTI; reverse transcriptase.The use of highly active antiretroviral therapy (HAART) involving multidrug combinations has significantly reduced the death rates of HIV-1 infected individuals receiving such treatment [1]. Inhibitors of the HIV-1 reverse transcriptase (RT) constitute a cornerstone in this therapy and are commonly used in combination with inhibitors of the HIV-1 protease. The RT inhibitors belong to two classes, the nucleoside inhibitors and the non-nucleoside inhibitors (NNRTI). Whereas the NRTIs are nucleoside analogues with chain-terminating properties and affinity to active site residues, the NNRTIs include a wide range of series of chemical compounds characterized by noncompetitive binding to an allosteric site some 10 Å away from the active site. Structural comparison of RT in complex with template/primer and NNRTIs together with native RT complexes have shown that the NNRTIs inhibit the polymerase activity through long-range and short-range structural distortions in several of the RT subdomains. The distortions involve repositioning of residues in the nonnucleoside binding pocket (NNIBP) that impose steric impediments on the thumb subdomain flexibility forcing it to remain in the ...

show abstract

Section: A T E R I a L S A N D M E T H O D Smentioning

confidence: 99%

“…The three NNRTIs in this study, Efavirenz, MSC194, and PNU142721, were tested in HIV-RT enzyme assays with wild-type RT and the K103N mutant [20]. The IC 50 values from these assays are presented in Table 2.…”

Section: Antiviral Activitymentioning

confidence: 99%

Structural basis for the inhibitory efficacy of efavirenz (DMP‐266), MSC194 and PNU142721 towards the HIV‐1 RT K103N mutant

Lindberg

Sigurðsson

Löwgren

et al. 2002

European Journal of Biochemistry

135

View full text Add to dashboard Cite

show abstract

“…For cloned HIV-1 RT, the assay was as described (36). Using extract from Escherichia coli without RT expression, incorporation of [3H]dGTP was =2000 cpm as trichloroacetic acid-precipitable material per 50 ,ul of assay mixture.…”

mentioning

confidence: 99%

“…Single-stranded DNA was isolated from the plasmid pRT.BS (36), containing the HIV-1(IIIB) RT sequence. Oligonucleoside-directed sitespecific mutagenesis was done according to the method by Sayers et al (37).…”

mentioning

confidence: 99%

Human immunodeficiency virus type 1 (HIV-1) strains selected for resistance against the HIV-1-specific [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-pentofurano syl (TSAO) nucleoside analogues retain sensitivity to HIV-1-specific nonnucleoside inhibitors.

Balzarini

Karlsson

Vandamme

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We recently reported that a newly discovered class of nucleoside analogues--[2',5'-bis-O-(tert-butyldimethylsilyl)- 3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D - pentofuranosyl derivatives of pyrimidines and purines (designated TSAO)--are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) and targeted at the nonsubstrate binding site of HIV-1 reverse transcriptase (RT). We now find that HIV-1 strains selected for resistance against three different TSAO nucleoside derivatives retain sensitivity to the other HIV-1-specific nonnucleoside derivatives (tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine, nevirapine, and pyridinone L697,661, as well as to the nucleoside analogues 3'-azido-3'-deoxythymidine, ddI, ddC, and 9-(2-phosphonylmethoxyethyl)adenine. Pol gene nucleotide sequence analysis of the TSAO-resistant and -sensitive HIV-1 strains revealed a single amino acid substitution at position 138 (Glu-->Lys) in the RT of all TSAO-resistant HIV-1 strains. HIV-1 RT in which the Glu-138-->Lys substitution was introduced by site-directed mutagenesis and expressed in Escherichia coli could not be purified because of rapid degradation. However, HIV-1 RT containing the Glu-138-->Arg substitution was stable. It lost its sensitivity to the TSAO nucleosides but not to the other HIV-1-specific RT inhibitors (i.e., TIBO and pyridinone). Our findings point to a specific interaction of the 4''-amino group on the 3'-spiro-substituted ribose moiety of the TSAO nucleosides with the carboxylic acid group of glutamic acid at position 138 of HIV-1 RT.

show abstract

“…Mutations in this region result in decreased catalytic efficiency and different kinetic properties (Bacolla et el., 1993;Zhang et et., 1993Zhang et et., , 1994. The altered inhibitory mechanism from non-competitive to mixed inhibition with regard to deoxynucleoside triphosphates by 9-CI-TIBO (Debyser et al, 1993), trovirdine (Zhang et al, 1995) and other non-nucleoside RT inhibitors (Byrnes et al, 1994) has also been documented for RT mutants.…”

Section: Synergy Of Trovirdine Combinations Against Hiv-1 225mentioning

confidence: 99%

Synergistic Inhibition of HIV-1 Reverse Transcriptase and HIV-1 Replication by Combining Trovirdine with AZT, ddl and ddCin Vitro

Zhang

Vrang

Rydergård

et al. 1996

Antivir Chem Chemother

Self Cite

View full text Add to dashboard Cite

SummaryTrovirdine (LY300046·HCI) is a potent and selective non-nucleoside human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor (Ahgren et al., Antimicrob Ag Chemother 39: 1329. Combinations of trovirdine with other RT inhibitors, AZT, ddC, ddl and their triphosphates, were studied as well as the pyrophosphate analogue PFA in both cell-free HIV-1 polymerase assays and HIV-1-infected MT-4 cell cultures. Synergistic effects and weak synergism were observed both using RT and HIV-1-infected cells and using different HIV-1 RT mutants and HIV-1 drug-resistant variants known to be resistant to the inhibitory effects of trovirdine. The best combination with substantial synergism was ddC-TP and trovirdine at a 20:1 molar ratio combination in a cell-free enzyme assay. This combination showed the weak synergy in MT-4 cells. Synergism was judged by the median-effect method. The inhibitory effect of trovirdine was independent of increased concentrations of AZT triphosphate and ddC triphosphate implying that trovirdine acts in a mutually exclusive manner with AZT-TP and ddC-TP as determined by the Dixon plot. The combination effects were expressed by the combination index (CI) using end points of 50%, 70% and 90% inhibition of HIV-1 RT activity and HIV-1 replication in MT-4 cells.

show abstract

Characterization of HIV Reverse Transcriptases with Tyr181→Cys and Leu100→lle Mutations

Cited by 17 publications

References 36 publications

Structural basis for the inhibitory efficacy of efavirenz (DMP‐266), MSC194 and PNU142721 towards the HIV‐1 RT K103N mutant

Structural basis for the inhibitory efficacy of efavirenz (DMP‐266), MSC194 and PNU142721 towards the HIV‐1 RT K103N mutant

Synergistic Inhibition of HIV-1 Reverse Transcriptase and HIV-1 Replication by Combining Trovirdine with AZT, ddl and ddCin Vitro

Contact Info

Product

Resources

About