scholarly journals Structure-Activity Analysis of Vinylogous Urea Inhibitors of Human Immunodeficiency Virus-Encoded Ribonuclease H

2010 ◽  
Vol 54 (9) ◽  
pp. 3913-3921 ◽  
Author(s):  
Suhman Chung ◽  
Michaela Wendeler ◽  
Jason W. Rausch ◽  
Greg Beilhartz ◽  
Matthias Gotte ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Single Molecule ◽  
Thiophene Ring ◽  
Rnase H ◽  
Single Molecule Spectroscopy ◽  
Inhibitor Binding ◽  
Structure Activity ◽  
Immunodeficiency Virus ◽  
Definition Of ◽  
Hiv 1

ABSTRACT Vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (compounds 1 and 2, respectively) were recently identified to be modestly potent inhibitors of the RNase H activity of HIV-1 and HIV-2 reverse transcriptase (RT). Both compounds shared a 3-CONH2-substituted thiophene ring but were otherwise structurally unrelated, which prevented a precise definition of the pharmacophore. We have therefore examined a larger series of vinylogous ureas carrying amide, amine, and cycloalkane modifications of the thiophene ring of compound 1. While cycloheptane- and cyclohexane-substituted derivatives retained potency, cyclopentane and cyclooctane substitutions eliminated activity. In the presence of a cycloheptane ring, modifying the 2-NH2 or 3-CONH2 functions decreased the potency. With respect to compound 2, vinylogous ureas whose dimethylthiophene ring contained modifications of the 2-NH2 and 3-CONH2 functions were investigated. 2-NH2-modified analogs displayed potency equivalent to or enhanced over that of compound 2, the most active of which, compound 16, reflected intramolecular cyclization of the 2-NH2 and 3-CONH2 groups. Molecular modeling was used to define an inhibitor binding site in the p51 thumb subdomain, suggesting that an interaction with the catalytically conserved His539 of the p66 RNase H domain could underlie inhibition of RNase H activity. Collectively, our data indicate that multiple functional groups of vinylogous ureas contribute to their potencies as RNase H inhibitors. Finally, single-molecule spectroscopy indicates that vinylogous ureas have the property of altering the reverse transcriptase orientation on a model RNA-DNA hybrid mimicking initiation plus-strand DNA synthesis.

scholarly journals Selection of Mutations in the Connection and RNase H Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Increase Resistance to 3′-Azido-3′-Dideoxythymidine

2007 ◽  
Vol 81 (15) ◽  
pp. 7852-7859 ◽  
Author(s):  
Jessica H. Brehm ◽  
Dianna Koontz ◽  
Jeffrey D. Meteer ◽  
Vinay Pathak ◽  
Nicolas Sluis-Cremer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Rnase H ◽  
Cross Resistance ◽  
Polymerase Domain ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3′-azido-3′-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1LAI or AZT-resistant HIV-1LAI (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1LAI were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance (∼16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance ∼10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.

Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase by Degradation Products of Ceftazidime

1997 ◽  
Vol 8 (4) ◽  
pp. 353-362 ◽  
Author(s):  
SW Baertschi ◽  
AS Cantrell ◽  
MT Kuhfeld ◽  
U Lorenz ◽  
DB Boyd ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Degradation Products ◽  
Rnase H ◽  
Immunodeficiency Virus ◽  
Hiv 1

Previous work by Hafkemeyer et al. (1991) [ Nucleic Acids Research19: 4059–4065] indicated that a degradation product of ceftazidime, termed HP 0.35, was active against the RNase H activity of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptase (RT) in vitro. Attempting to repeat these results, we isolated HP 0.35 from an aqueous degradation of ceftazidime and, after careful purification, we found HP 0.35 to be essentially inactive against both the polymerase and RNase H domains of HIV-1 RT (IC50 of >100 μg mL−1). During the investigation we discovered that polymeric degradation products of ceftazidime inhibited both the polymerase and, to a greater extent, the RNase H activities of HIV-1 RT in vitro (IC50 approximately 0.1 and 0.01 μg mL−1, respectively). Subjecting HP 0.35 to conditions under which it could polymerize induced inhibitory activity similar to that of the polymeric ceftazidime degradation products. It is proposed that the previously reported activity of HP 0.35 may have resulted from the presence of low levels of polymeric material either from incomplete purification or from polymerization of HP 0.35 during storage or in vitro testing.

scholarly journals Mutations in the U5 Region Adjacent to the Primer Binding Site Affect tRNA Cleavage by Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

2007 ◽  
Vol 82 (2) ◽  
pp. 719-727 ◽  
Author(s):  
Jangsuk Oh ◽  
Mary Jane McWilliams ◽  
John G. Julias ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Rnase H ◽  
Primer Binding Site ◽  
Cleavage Specificity ◽  
Immunodeficiency Virus ◽  
Trna Primer ◽  
Hiv 1

ABSTRACT In retroviruses, the first nucleotide added to the tRNA primer defines the end of the U5 region in the right long terminal repeat, and the subsequent removal of this tRNA primer by RNase H exactly defines the U5 end of the linear double-stranded DNA. In most retroviruses, the entire tRNA is removed by RNase H cleavage at the RNA/DNA junction. However, the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase cleaves the tRNA 1 nucleotide from the RNA/DNA junction at the U5/primer binding site (PBS) junction, which leaves an rA residue at the U5 terminus. We made sequence changes at the end of the U5 region adjacent to the PBS in HIV-1 to determine whether such changes affect the specificity of tRNA primer cleavage by RNase H. In some of the mutants, RNase H usually removed the entire tRNA, showing that the cleavage specificity was shifted by 1 nucleotide. This result suggests that the tRNA cleavage specificity of the HIV-1 RNase domain H depends on sequences in U5.

Structure-Activity Relationship Studies on Potential Non-Nucleoside DABO-Like Inhibitors of HIV-1 Reverse Transcriptase

2000 ◽  
Vol 11 (2) ◽  
pp. 117-133 ◽  
Author(s):  
Roberta Costi ◽  
Roberto Di Santo ◽  
Marino Artico ◽  
Silvio Massa ◽  
Antonio Lavecchia ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Structure Activity Relationship ◽  
Activity Relationship ◽  
Enzyme Assays ◽  
Structure Activity ◽  
Immunodeficiency Virus ◽  
Anti Hiv ◽  
Hiv 1

Using 2,6-dichloro-4-aminopyrimidine, a number of uracil and cytosine derivatives with both arylthio and alkoxy moieties were prepared. These novel pyrimidines share chemical similarities with DABOs and HEPTs, two classes of nonnucleoside human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitors (NNRTIs), which have been widely studied of late. All new derivatives were tested in MT-4 cells to explore their potential in vivo anti-HIV activity. Like other NNRTIs, they selectively inhibit HIV-1 but not HIV- 2. The majority of test derivatives were found to have low potency and were sometimes more cytotoxic than zidovudine and emivirine (formerly MKC-442), used here as reference drugs. Uracil and cytosine derivatives bearing a sec-butoxy chain and a methyl-substituted benzenesulphonyl moiety were the most potent. Enzyme assays proved that these derivatives target RT. Structure-activity relationship studies established a correlation between the anti-HIV-1 activity and the meta substitution on the phenyl ring; furthermore, oxidation of sulphide to sulphone significantly increased the potency of certain derivatives.

scholarly journals Interaction between Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Proteins

2004 ◽  
Vol 78 (10) ◽  
pp. 5056-5067 ◽  
Author(s):  
Eric A. Hehl ◽  
Pheroze Joshi ◽  
Ganjam V. Kalpana ◽  
Vinayaka R. Prasad
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rnase H ◽  
Binding Domain ◽  
Immunodeficiency Virus ◽  
Carboxy Terminal ◽  
Hiv 1

ABSTRACT Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses. It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X. Wu, H. Liu, H. Xiao, J. A. Conway, E. Hehl, G. V. Kalpana, V. R. Prasad, and J. C. Kappes, J. Virol. 73:2126-2135, 1999). We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function. Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN. An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction. Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT. Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain. The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment. This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction. The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3′ processing and joining reactions in the presence of both proteins. Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold. Thus, a functional interaction between these two viral enzymes may occur during viral replication.

scholarly journals Antiretroviral Drug Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Increase Template-Switching Frequency

2004 ◽  
Vol 78 (16) ◽  
pp. 8761-8770 ◽  
Author(s):  
Galina N. Nikolenko ◽  
Evguenia S. Svarovskaia ◽  
Krista A. Delviks ◽  
Vinay K. Pathak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rnase H ◽  
Switching Frequency ◽  
Template Switching ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Template-switching events during reverse transcription are necessary for completion of retroviral replication and recombination. Structural determinants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that influence its template-switching frequency are not known. To identify determinants of HIV-1 RT that affect the frequency of template switching, we developed an in vivo assay in which RT template-switching events during viral replication resulted in functional reconstitution of the green fluorescent protein gene. A survey of single amino acid substitutions near the polymerase active site or deoxynucleoside triphosphate-binding site of HIV-1 RT indicated that several substitutions increased the rate of RT template switching. Several mutations associated with resistance to antiviral nucleoside analogs (K65R, L74V, E89G, Q151N, and M184I) dramatically increased RT template-switching frequencies by two- to sixfold in a single replication cycle. In contrast, substitutions in the RNase H domain (H539N, D549N) decreased the frequency of RT template switching by twofold. Depletion of intracellular nucleotide pools by hydroxyurea treatment of cells used as targets for infection resulted in a 1.8-fold increase in the frequency of RT template switching. These results indicate that the dynamic steady state between polymerase and RNase H activities is an important determinant of HIV-1 RT template switching and establish that HIV-1 recombination occurs by the previously described dynamic copy choice mechanism. These results also indicate that mutations conferring resistance to antiviral drugs can increase the frequency of RT template switching and may influence the rate of retroviral recombination and viral evolution.

scholarly journals Mutation of Amino Acids in the Connection Domain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Contact the Template-Primer Affects RNase H Activity

2003 ◽  
Vol 77 (15) ◽  
pp. 8548-8554 ◽  
Author(s):  
John G. Julias ◽  
Mary Jane McWilliams ◽  
Stefan G. Sarafianos ◽  
W. Gregory Alvord ◽  
Edward Arnold ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Rnase H ◽  
Viral Dna ◽  
Viral Dnas ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The crystal structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in a complex with an RNA-DNA template-primer identified amino acids in the connection domain that make specific contacts with the nucleic acid. We analyzed the effects of mutations in these amino acids by using a one-round HIV-1 vector. Mutations in amino acids in the connection domain generally had small effects on virus titers. To determine whether the mutations affected the level of RNase H activity or the specificity of RNase H cleavage, we used the two-long-terminal-repeat circle junction as a surrogate for the ends of linear viral DNA; specific RNase H cleavages determine the ends of the viral DNA. Several of the mutations in the connection domain affected the frequency of the generation of viral DNAs with aberrant ends. The mutation H361A had the largest effect on the titer and on the generation of DNAs with aberrant ends. H361 contacts the phosphate backbone of the nucleic acid in the same location as amino acid Y501 in the RNase H primer grip. Mutations at Y501 have been shown to decrease the virus titer and affect the specificity of RNase H cleavage. H361A affected the frequency of the generation of linear viral DNAs with aberrant ends, but in general the connection domain mutations had subtle effects on the efficiency of RNase H cleavage. The results of this study suggest that, in addition to its primary role in linking the polymerase and RNase H domains, the connection subdomain has a modest role in binding and positioning the nucleic acid.

scholarly journals Structure of a Dihydroxycoumarin Active-Site Inhibitor in Complex with the RNase H Domain of HIV-1 Reverse Transcriptase and Structure–Activity Analysis of Inhibitor Analogs

2014 ◽  
Vol 426 (14) ◽  
pp. 2617-2631 ◽  
Author(s):  
Daniel M. Himmel ◽  
Nataliya S. Myshakina ◽  
Tatiana Ilina ◽  
Alexander Van Ry ◽  
William C. Ho ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Activity Analysis ◽  
Rnase H ◽  
Structure Activity ◽  
Hiv 1

scholarly journals Reverse transcriptase backbone can alter the polymerization and RNase activities of non-nucleoside reverse transcriptase mutants K101E+G190S

2013 ◽  
Vol 94 (10) ◽  
pp. 2297-2308
Author(s):  
Jiong Wang ◽  
Dongge Li ◽  
Robert A. Bambara ◽  
Carrie Dykes
Keyword(s):  
Reverse Transcriptase ◽  
Resistant Mutant ◽  
Rnase H ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Virus Growth ◽  
Immunodeficiency Virus ◽  
Drug Resistant Mutant ◽  
Replication Fitness ◽  
Hiv 1

Previous work by our group showed that human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing non-nucleoside RT inhibitor (NNRTI) drug resistance mutations has defects in RNase H activity as well as reduced amounts of RT protein in virions. These deficits correlate with replication fitness in the absence of NNRTIs. Viruses with the mutant combination K101E+G190S replicated better in the presence of NNRTIs than in the absence of drug. Stimulation of virus growth by NNRTIs occurred during the early steps of the virus life cycle and was modulated by the RT backbone sequence in which the resistance mutations arose. We wanted to determine what effects RT backbone sequence would have on RT content and polymerization and RNase H activities in the absence of NNRTIs. We compared a NL4-3 RT with K101E+G190S to a patient-isolate RT sequence D10 with K101E+G190S. We show here that, unlike the NL4-3 backbone, the D10 backbone sequence decreased the RNA-dependent DNA polymerization activity of purified recombinant RT compared to WT. In contrast, RTs with the D10 backbone had increased RNase H activity compared to WT and K101E+G190S in the NL4-3 backbone. D10 virions also had increased amounts of RT compared to K101E+G190S in the NL4-3 backbone. We conclude that the backbone sequence of RT can alter the activities of the NNRTI drug-resistant mutant K101E+G190S, and that identification of the amino acids responsible will aid in understanding the mechanism by which NNRTI drug-resistant mutants alter fitness and NNRTIs stimulate HIV-1 virus replication.

Sign in / Sign up

Export Citation Format

Share Document