scholarly journals Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Paul L. Boyer ◽  
Steven J. Smith ◽  
Xue Zhi Zhao ◽  
Kalyan Das ◽  
Kevin Gruber ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Nucleoside Analogs ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv Replication ◽  
Content Type ◽  
Rnase Hi ◽  
Hiv 1

ABSTRACT We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds. IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

scholarly journals Expression of an Mg2+-Dependent HIV-1 RNase H Construct for Drug Screening

2011 ◽  
Vol 55 (10) ◽  
pp. 4735-4741 ◽  
Author(s):  
Richard V. Farias ◽  
Deborah A. Vargas ◽  
Andres E. Castillo ◽  
Beatriz Valenzuela ◽  
Marie L. Coté ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rnase H ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Wild Type ◽  
Content Type ◽  
Dependent Activity ◽  
Rnase Hi ◽  
Single Polypeptide ◽  
Hiv 1

ABSTRACTA single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg2+-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of anEscherichia colistrain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg2+-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similarKms for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

scholarly journals Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Paul L. Boyer ◽  
Kevin Melody ◽  
Steven J. Smith ◽  
Linda L. Dunn ◽  
Chris Kline ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Active Site ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Hydrophobic Region ◽  
Nnrti Resistance ◽  
Hiv 1

ABSTRACTTwo mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the “primer grip” region and could be influenced by the M230I mutation.IMPORTANCEAlthough antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

Recombinant HIV-1 Nucleocapsid Protein Accelerates HIV-1 Reverse Transcriptase Catalyzed DNA Strand Transfer Reactions and Modulates RNase H Activity

Biochemistry ◽  
1994 ◽  
Vol 33 (46) ◽  
pp. 13817-13823 ◽  
Author(s):  
James A. Peliska ◽  
Shankar Balasubramanian ◽  
David P. Giedroc ◽  
Stephen J. Benkovic
Keyword(s):  
Reverse Transcriptase ◽  
Nucleocapsid Protein ◽  
Rnase H ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Dna Strand ◽  
Hiv 1

scholarly journals Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

2000 ◽  
Vol 74 (7) ◽  
pp. 3245-3252 ◽  
Author(s):  
Susanne Werner ◽  
Birgitta M. Wöhrl
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Rous Sarcoma Virus ◽  
Active Sites ◽  
Polymerase Activity ◽  
Rnase H ◽  
Wild Type ◽  
Α Subunit ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The genes encoding the α (63-kDa) and β (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV αβ and αPol RTs within which one subunit was selectively mutated. When αβ heterodimers contained the Asp 181→Asn mutation in their β subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their α subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in αβ heterodimers mutated in the β subunit but was lost when the mutation was present in the α subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the α subunit of the heterodimeric RSV RT αβ.

scholarly journals Structure for an HIV-1 reverse transcriptase RNase H inhibitor bound at the active site

2008 ◽  
Vol 64 (a1) ◽  
pp. C348-C348
Author(s):  
D.M. Himmel ◽  
T.A. Pauly ◽  
J. Bauman ◽  
C. Dharia ◽  
A.D. Clark ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Rnase H ◽  
Hiv 1

A Review on Pharmacokinetics properties of antiretroviral drugs to treat HIV-1 infections

2020 ◽  
Vol 16 ◽  
Author(s):  
Sanjeev Kumar Singh ◽  
Mohd. Aqueel Khan ◽  
Krishna Kant Gupta
Keyword(s):  
Reverse Transcriptase ◽  
Antiretroviral Drugs ◽  
Hiv Treatment ◽  
Strand Transfer ◽  
Reverse Transcriptase Inhibitors ◽  
Hiv Replication ◽  
Health Crisis ◽  
Pharmacokinetic Properties ◽  
Approved Drugs ◽  
Hiv 1

Background: The HIV-1 pandemic is undoubtedly the major public-health crisis of our time. The extensive research on HIV has deepened our understanding of its pathogenesis and transmission dynamics. Some new entity molecules have been approved by the FDA for HIV treatment but till now protective vaccine remains elusive. Scientists are targeting many important proteins of HIV-1; gp41, gp120, CCR5 coreceptor, integrase, reverse transcriptase and protease. Few compounds are used as nucleotide analogues to stop HIV replication. Altogether, these compounds and their derivatives specifically block HIV entry and DNA replication. Using ADMET studies, people are working on these compounds to reduce toxicity and increase potency. Objective: Our main aim is to discuss the Pharmacokinetics properties of 23 important FDA antiretroviral drugs used for the treatment of HIV-1 infections. Methods: We have searched literature related to pharmacokinetics properties in PubMed, Google Scholar search engine. Conclusion: Here, we have reviewed the pharmacokinetic properties such as absorption, bioavailability, distribution, metabolism, and excretion, of important 23 FDA approved drugs. The drugs namely Fuzeon, Selzentry, Complera, Epivir, Retrovir, Emtriva, Ziagen, Edurant, Intelence, Pifeltro, Sustiva, Viramune, Isentress, Genvoya, Tivicay, Reyataz, Prezista, Lexiva, Invirase, Aptivus etc. are classified into five major classes: fusion inhibitors, Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), Integrase Strand transfer inhibitors (INSTIs) and Protease inhibitors (PIs). This Review may helpful for the future development of potent antiretroviral drugs with improved pharmacokinetic properties.

scholarly journals Structure of HIV-1 Reverse Transcriptase with the Inhibitor β-Thujaplicinol Bound at the RNase H Active Site

Structure ◽  
2009 ◽  
Vol 17 (12) ◽  
pp. 1625-1635 ◽  
Author(s):  
Daniel M. Himmel ◽  
Karen A. Maegley ◽  
Tom A. Pauly ◽  
Joseph D. Bauman ◽  
Kalyan Das ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Rnase H ◽  
Hiv 1

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase, RNase H, and Integrase Activities by Hydroxytropolones

2005 ◽  
Vol 49 (12) ◽  
pp. 4884-4894 ◽  
Author(s):  
Joël Didierjean ◽  
Catherine Isel ◽  
Flore Querré ◽  
Jean-François Mouscadet ◽  
Anne-Marie Aubertin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Metal Ions ◽  
Dna Polymerase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Rnase H ◽  
Strand Transfer ◽  
Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type I reverse transcriptase (RT) possesses distinct DNA polymerase and RNase H sites, whereas integrase (IN) uses the same active site to perform 3′-end processing and strand transfer of the proviral DNA. These four enzymatic activities are essential for viral replication and require metal ions. Two Mg2+ ions are present in the RT polymerase site, and one or two Mg2+ ions are required for the catalytic activities of RNase H and IN. We tested the possibility of inhibition of the RT polymerase and RNase H as well as the IN 3′-end processing and transfer activities of purified enzymes by a series of 3,7-dihydroxytropolones designed to target two Mg2+ ions separated by ∼3.7 Å. The RT polymerase and IN 3′ processing and strand transfer activities were inhibited at submicromolar concentrations, while the RNase H activity was inhibited in the low micromolar range. In all cases, the lack of inhibition by tropolones and O-methylated 3,7-dihydroxytropolones was consistent with the active molecules binding the metal ions in the active site. In addition, inhibition of the DNA polymerase activity was shown to depend on the Mg2+ concentration. Furthermore, selective inhibitors were identified for several of the activities tested, leaving some potential for design of improved inhibitors. However, all tested compounds exhibited cellular toxicity that presently limits their applications.

scholarly journals HIV-1 Reverse Transcriptase Still Remains a New Drug Target: Structure, Function, Classical Inhibitors, and New Inhibitors with Innovative Mechanisms of Actions

2012 ◽  
Vol 2012 ◽  
pp. 1-23 ◽  
Author(s):  
Francesca Esposito ◽  
Angela Corona ◽  
Enzo Tramontano
Keyword(s):  
Reverse Transcriptase ◽  
Drug Target ◽  
Polymerase Activity ◽  
Rnase H ◽  
Primary Target ◽  
Lethal Mutagenesis ◽  
Rt Inhibitors ◽  
Process Characteristic ◽  
Hiv 1 ◽  
Chain Terminators

During the retrotranscription process, characteristic of all retroviruses, the viral ssRNA genome is converted into integration-competent dsDNA. This process is accomplished by the virus-coded reverse transcriptase (RT) protein, which is a primary target in the current treatments for HIV-1 infection. In particular, in the approved therapeutic regimens two classes of drugs target RT, namely, nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). Both classes inhibit the RT-associated polymerase activity: the NRTIs compete with the natural dNTP substrate and act as chain terminators, while the NNRTIs bind to an allosteric pocket and inhibit polymerization noncompetitively. In addition to these two classes, other RT inhibitors (RTIs) that target RT by distinct mechanisms have been identified and are currently under development. These include translocation-defective RTIs, delayed chain terminators RTIs, lethal mutagenesis RTIs, dinucleotide tetraphosphates, nucleotide-competing RTIs, pyrophosphate analogs, RT-associated RNase H function inhibitors, and dual activities inhibitors. This paper describes the HIV-1 RT function and molecular structure, illustrates the currently approved RTIs, and focuses on the mechanisms of action of the newer classes of RTIs.

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