Download or read book Kinetic Analysis of HIV 1 Reverse Transcriptase in the Presence of Non nucleoside Inhibitors written by Louise Zhiying Wang and published by . This book was released on 2004 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Kinetic Studies of HIV 1 Reverse Transcriptase Nucleotide Selectivity Drug Resistance and RNase H Activity written by An Li (Ph. D. in biochemistry) and published by . This book was released on 2016 with total page 456 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mechanisms of nucleotide selectivity and drug resistance by HIV-1 Reverse Transcriptase (HIVRT) were examined using rapid kinetic methods and global data fitting. Thymidine analog resistance mutations provide only two-fold discrimination against the incorporation of AZT-triphosphate; therefore, it is generally believed that resistance arises from nucleotide excision, where ATP reacts with the 3’ terminal base to produce a dinucleotide tetraphosphate and a 3’-OH terminated primer capable of subsequent extension. Single turnover kinetic analysis with global data fitting revealed the intrinsic rate and equilibrium constants governing each step leading to resistance to chain terminators. Our data suggested the net resistance to AZT arises from nearly equal contributions involving discrimination during incorporation (2x), enhanced ATP-dependent excision (2-5x), reduced binding of the next correct nucleotide (2.6-5x) and more favored binding of the DNA primer in the N-site (5-20x). Chemistry is generally the only rate-limiting step for product formation during DNA polymerization with a DNA template, but with an RNA template we show that pyrophosphate (PPi) release was rate-limiting. Due to the slow PPi release step, the rate of reversal of chemistry could also be determined affording the first measurement of the equilibrium constants governing polymerization and the first complete free energy profile for HIVRT. Although PPi release is rate-limiting, nucleotide binding remains as the specificity-determining step. However, PPi release becomes exceedingly slow following mis-incorporation and thereby contributes to the specificity constant. Our data demonstrate that the fidelity of HIVRT has been underestimated by >20-fold in the past 20 years since the slow PPi release has been overlooked. The rate-limiting PPi release allows synchronization and coordination of the polymerase and RNase-H activities. Studies were undertaken to examine proposed coordination between the polymerase and RNase-H activities. Direct, simultaneous measurement of the two activities established a mechanism by which polymerization and RNase-H activities are coordinated by working independently at comparable rates, but with different efficiencies. In contrast to polymerization, RNase-H requires ~4–6 base pairs extending beyond the active site for optimal reactivity, providing fast but infrequent cleavage events. Consequently, the polymerase and RNase-H activities are seamlessly coordinated without any direct communication between the two sites.
Download or read book Human Immunodeficiency Virus Reverse Transcriptase written by Stuart LeGrice and published by Springer Science & Business Media. This book was released on 2013-07-23 with total page 358 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Reverse Transcriptase (RT) of Human Immunodeficiency Virus Type 1 (HIV-1) arguably ranks amongst one of the most extensively studied retroviral enzymes. Heterologous expression and purification of HIV-1 RT in the early eighties, approval of the first nucleoside analogue RT inhibitor (NRTI) in 1987, discovery of resistance to RT inhibitors, approval of the first non-nucleoside analogue RT inhibitor (NNRTI) in 1996 and the various crystal structures of RT with and without bound substrate(s) and/or inhibitors represent only a few of the important milestones that describe the a bench-to-bedside success in the continuing effort to combat HIV-1 infection and its consequences. Nucleoside and nonnucleoside RT inhibitors remain important components in frequently used drug regimens to treat the infection. RT inhibitors also play important roles in recently validated strategies to prevent transmission of the virus. The relevance of HIV-1 RT as a drug target has simultaneously triggered interest in basic research studies aimed at providing a more detailed understanding of interactions between proteins, nucleic acids, and small molecule ligands in general terms. In light of the ever-growing knowledge on structure and function of HIV-1 RT, this enzyme serves as a valuable “model system” in efforts to develop novel experimental tools and to explain biochemical processes. This monograph is designed to provide an overview of important aspects in past and current HIV-1 RT research, with focus on mechanistic aspects and translation of knowledge into drug discovery and development. The first section includes chapters with emphasis placed on the coordination of the RT-associated DNA polymerase and ribonuclease H (RNase H) activities. The second covers mechanisms of action and future perspectives associated with NRTIs and NNRTIs, while the third section includes chapters focusing on novel strategies to target the RT enzyme. Chapters of the final part are intended to discuss mechanisms involved in HIV variability and the development of drug resistance. We hope that these contributions will stimulate interest, and encourage research aimed at the development of novel RT inhibitors. The lack of bona fide RNase H inhibitors with potent antiviral activity provides an example for challenges and opportunities in the field.
Download or read book Reverse Transcriptase Inhibitors in HIV AIDS Therapy written by Gail Skowron and published by Springer Science & Business Media. This book was released on 2007-11-10 with total page 536 pages. Available in PDF, EPUB and Kindle. Book excerpt: A magisterial survey of all aspects of the reverse transcriptase inhibitors (RTIs) used to treat HIV/AIDS, including drug discovery, pharmacology, development of drug resistance, toxicity, and prevention of mother-to-child transmission of HIV/AIDS. The authors synthesize our current understanding of the role of reverse transcriptase in the viral life cycle, describe the discovery and development of eight nucleoside and nucleotide analogs that represent milestones in treatment history, and thoroughly discuss the question of toxicity and resistance to this class of drugs. They also address three non-nucleoside RTIs and their pharmacokinetics and comparative clinical efficacy, new RTIs currently under development, and the impact of approved agents on treatment, in general, and on vertical transmission in the developing world.
Download or read book Effects of Antiretroviral Drugs on the Translocation Status of Human Immunodeficiency Virus Type 1 Reverse Transcriptase written by Bruno Marchand and published by . This book was released on 2007 with total page 239 pages. Available in PDF, EPUB and Kindle. Book excerpt: "The reverse transcriptase of the Human Immunodeficiency Virus type 1 (HIV-1) is responsible for transcribing the viral single-stranded RNA genome into double-stranded DNA. Due to its vital role in the viral life cycle, it is often targeted in the treatment of HIV-1 infected patients. Inhibitors that block the reverse transcriptase enzyme were shown to interfere with nucleotide incorporation. At the end of a nucleotide incorporation cycle, the newly incorporated substrate is still located in the nucleotide binding site. In order to continue the elongation of the DNA chain, the enzyme has to move by one nucleotide relative to the nucleic acid template to free the nucleotide binding site. This movement is referred to as translocation. Translocation is extremely rapid and is therefore kinetically invisible. In order to study the mechanism of translocation, I developed site-specific footprinting assays with a resolution of a single nucleotide, which allowed us to detect the precise positioning of the reverse transcriptase on a DNA template. Using these techniques, in combination with kinetic analysis as well as other enzymatic assays, I studied the translocational status of the reverse transcriptase in various conditions, and concluded that this enzyme translocates according to a 0́−Brownian ratchet model0́+. In this model, the enzyme oscillates between pre- and post-translocated positions and the nucleotide substrate traps the post-translocational state. I identified important factors that affect the translocational equilibrium: the sequence of the nucleic acid template, the nature of the nucleotide at the 30́9 end of the primer, the presence of mismatches in the double-stranded nucleic acid substrate, the presence of nucleotides, the presence of inhibitors, the nature of these inhibitors, the presence of drug resistance conferring mutations and the temperature are parameters that have all been shown to modify the translocational equilibrium of the reverse" --
Download or read book Structural Analysis of HIV 1 Reverse Transcriptase Complexed with Various Nonnucleoside Inhibitors Using X ray Crystallography written by Wanyi Zhang and published by . This book was released on 1998 with total page 262 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Design Synthesis and Biological Testing of Novel Non nucleoside HIV 1 Reverse Transcriptase Inhibitors written by Jitendra D. Belani and published by . This book was released on 2002 with total page 358 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book The Use of Non nucleoside Reverse Transcriptase Inhibitors in the Treatment of HIV 1 Infection written by Franciscus Cornelis Maria Leth and published by . This book was released on 2005 with total page 215 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Biochemical Characterization of Clade B and Non B HIV 1 Reverse Transcriptase written by Jacqueline A. Flores and published by . This book was released on 2017 with total page 43 pages. Available in PDF, EPUB and Kindle. Book excerpt: Two classes of drugs: nucleos(t)ide reverse transcriptase inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) target HIV-1 RT, an enzyme required for HIV replication. The first generation NNRTIs (nevirapine and efavirenz) have been extensively used worldwide. However, they have a low genetic barrier for resistance. The second generation NNRTI, Rilpivirine (RPV) is effective against viruses that are resistant to nevirapine and efavirenz. RPV has not been used against subtype C HIV-1, which accounts for ~50% of all infections. Recent clinical trials have shown that patients with subtype C are more likely to fail RPV-based treatment. However, the mechanism of RPV-failure has not been determined. To get insight into RPV-failure, RTs from patient isolates were cloned and biochemically characterized using pre-steady state kinetics to determine: 1) the impact of genetic variation on HIV-1 replication, and 2) the effect of a known RPVresistance mutation (p66[subscript E138K-M184I]/p51[subscript E138K]) on the initiation of reverse transcription, the first step of HIV replication. The results showed that subtype C HIV-1 RT has lower DNA and dNTP binding affinities, and RPV binding affinity than subtype B HIV-1 RT. The p66[subscript E138K-M184I]/p51[subscript E138K] mutation was found to cause rilpivirine to dissociate from p66[subscript E138KM184I]/p51[subscript E138K] RT 3x faster than wild type RT at initiation - suggesting that RPV may not be an optimal choice for that subtype C infections.
Download or read book A Kinetic Analysis of Human Immunodeficiency Virus Reverse Transcriptase written by Shawn Patrick Zinnen and published by . This book was released on 1994 with total page 258 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Study of the Effects of Mutations in HIV 1 Reverse Transcriptase on Resistance Against Non nucleoside Reverse Transcriptase Inhibitors written by Heidi Pelemans and published by . This book was released on 2001 with total page 129 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Antimicrobial Drug Resistance written by Douglas L. Mayers and published by Springer. This book was released on 2017-07-07 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The two volumes included in Antimicrobial Drug Resistance, Second Edition is an updated, comprehensive and multidisciplinary reference covering the area of antimicrobial drug resistance in bacteria, fungi, viruses, and parasites from basic science, clinical, and epidemiological perspectives. This newly revised compendium reviews the most current research and development on drug resistance while still providing the information in the accessible format of the first edition. The first volume, Antimicrobial Drug Resistance: Mechanisms of Drug Resistance, is dedicated to the biological basis of drug resistance and effective avenues for drug development. With the emergence of more drug-resistant organisms, the approach to dealing with the drug resistance problem must include the research of different aspects of the mechanisms of bacterial resistance and the dissemination of resistance genes as well as research utilizing new genomic information. These approaches will permit the design of novel strategies to develop new antibiotics and preserve the effectiveness of those currently available. The second volume, Antimicrobial Drug Resistance: Clinical and Epidemiological Aspects, is devoted to the clinical aspects of drug resistance. Although there is evidence that restricted use of a specific antibiotic can be followed by a decrease in drug resistance to that agent, drug resistance control is not easily achieved. Thus, the infectious diseases physician requires input from the clinical microbiologist, antimicrobial stewardship personnel, and infection control specialist to make informed choices for the effective management of various strains of drug-resistant pathogens in individual patients. This 2-volume set is an important reference for students in microbiology, infectious diseases physicians, medical students, basic scientists, drug development researchers, microbiologists, epidemiologists, and public health practitioners.
Download or read book The Use of Non nucleoside Reverse Transcriptase Inhibitors in the Treatment of HIV 1 Infection written by Frank van Leth and published by . This book was released on 2005 with total page 211 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book The Design of Non nucleoside Inhibitors of HIV 1 with Enhanced Interactions with Reverse Transcriptase written by Souzan Armstrong and published by . This book was released on 1997 with total page 324 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Characterization of HIV 1 Reverse Transcriptase Substrate Specificity by Conformationally Sensitive Fluorescence written by Matthew William Kellinger and published by . This book was released on 2010 with total page 390 pages. Available in PDF, EPUB and Kindle. Book excerpt: We have engineered a mutant of HIV Reverse Transcriptase that can be fluorescently labeled by covalent attachment of the environmentally sensitive fluorophore 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC). The result is a polymerase that is kinetically indistinguishable from the wild-type enzyme, but provides a signal to monitor changes in enzyme structure that result from conformational changes induced by substrate binding. Using this system, we have expanded the kinetic model governing nucleotide binding to include an enzymatic isomerization following initial nucleotide binding. In doing so, we define the role of induced-fit in nucleotide specificity and mismatch discrimination. Additionally, we have characterized the kinetics governing the specificity and discrimination of several widely administered Nucleotide Reverse Transcriptase Inhibitors (NRTI's) used to combat HIV infection including 3TC (Lamivudine), FTC (Emtricitabine), and AZT (Zidovudine) for the wild-type polymerase and mutants with clinical resistance to these compounds. Our findings resolve the apparent tighter binding of these inhibitor compounds compared to the correct nucleotide by showing that the affinity for the correct nucleotide is stronger than the inhibitors. The apparent weaker binding of the correct nucleotide is a result of a incomplete interpretation of binding data that fails to account for the importance of the reverse rate of the conformational change. The apparent Kd (Kd, app) measurements for correct nucleotide estimates Km rather than Kd because nucleotide binding does not reach equilibrium. The conformationally sensitive enzyme has also been used to characterize the kinetics governing DNA association. We show that DNA binding is governed by a two-step process where a fast initial association is followed by a second, slow isomerization that is off the pathway for nucleotide binding and incorporation. Finally, we have implemented single molecule techniques using fluorophore labeled nucleotides to study the effects of AZT incorporation on the DNA translocation dynamics of the polymerase. We find that primer termination with AZT results in DNA that fails to translocate, therefore occluding the next nucleotide from binding. This shift in translocation equilibrium exposes the newly formed phosphodiester bond to ATP- or pyrophosphate-mediated AZT excision; thereby rescuing productive polymerization. This finding represents the first kinetic measurement of DNA translocation by a polymerase.
Download or read book Binding of the Nonnucleoside Reverse Transcriptase Inhibitor Efavirenz to HIV 1 Reverse Transcriptase Monomers and Dimers written by Valerie Ann Braz and published by . This book was released on 2010 with total page 161 pages. Available in PDF, EPUB and Kindle. Book excerpt: Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was the first target of antiretroviral therapy in the treatment of AIDS. RT converts single-stranded viral RNA into double-stranded proviral DNA. The enzyme has two activities, DNA polymerase and RNase H. The biologically relevant form is a heterodimer composed of two subunits, p66 and p51. The subunits are products of the same gene and have identical N-terminal amino acid sequences; p51 lacks the C-terminal RNase H domain. In solution RT exists as a complex equilibrium mixture of p66/p51 heterodimer, p66/p66 and p51/p51 homodimers, and p66 and p51 monomers. Two classes of inhibitors have been developed and approved for clinical use, NRTIs and NNRTIs. NNRTIs are highly effective and relatively noncytotoxic small, amphiphilic, noncompetitive inhibitors that nestle into a hydrophobic pocket near the polymerase active site in the p66 subunit of RT. NNRTIs also have diverse effects on RT subunit dimerization; some enhance dimerization and others weaken dimerization. Efavirenz is an NNRTI capable of affecting several steps in HIV-1 reverse transcription and replication. This work reports two novel functions of the inhibitor: (1) efavirenz, and presumably also other NNRTIs, binds to monomeric forms of RT and (2) efavirenz is a slow binding inhibitor of heterodimer and monomers. In addition, the binding site on p66 and p51 monomers was identified. Five techniques were used to characterize the interaction of efavirenz to all species of RT; equilibrium dialysis, tryptophan fluorescence, native gel electrophoresis, and hydrogen-deuterium exchange and Fourier transform ion cyclotron resonance mass spectrometry. Although the biological significance of efavirenz binding to monomeric forms of RT is not known, this work suggests that monomeric forms of RT may be potential targets for HIV-1 therapeutics.
Download or read book Nucleic Acid Polymerases written by Katsuhiko S. Murakami and published by Springer Science & Business Media. This book was released on 2013-10-22 with total page 342 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides a review of the multitude of nucleic acid polymerases, including DNA and RNA polymerases from Archea, Bacteria and Eukaryota, mitochondrial and viral polymerases, and other specialized polymerases such as telomerase, template-independent terminal nucleotidyl transferase and RNA self-replication ribozyme. Although many books cover several different types of polymerases, no book so far has attempted to catalog all nucleic acid polymerases. The goal of this book is to be the top reference work for postgraduate students, postdocs, and principle investigators who study polymerases of all varieties. In other words, this book is for polymerase fans by polymerase fans. Nucleic acid polymerases play a fundamental role in genome replication, maintenance, gene expression and regulation. Throughout evolution these enzymes have been pivotal in transforming life towards RNA self-replicating systems as well as into more stable DNA genomes. These enzymes are generally extremely efficient and accurate in RNA transcription and DNA replication and share common kinetic and structural features. How catalysis can be so amazingly fast without loss of specificity is a question that has intrigued researchers for over 60 years. Certain specialized polymerases that play a critical role in cellular metabolism are used for diverse biotechnological applications and are therefore an essential tool for research.
