scholarly journals REverSe TRanscrIptase Chain Termination (RESTRICT) for Selective Measurement of Nucleotide Analogs Used in HIV Care and Prevention

2021 ◽  
Author(s):  
Ayokunle Oluwafemi Olanrewaju ◽  
Benjamin Sullivan ◽  
Alicia Gim ◽  
Derin Sevenler ◽  
Andrew Bender ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Antiretroviral Drugs ◽  
Chain Termination ◽  
Hiv Care ◽  
Therapeutic Drug ◽  
Hiv Reverse Transcriptase ◽  
Nucleotide Analogs ◽  
New Class ◽  
Drug Concentrations ◽  
Inhibition Of Dna Synthesis

Sufficient drug concentrations are required for efficacy of antiretroviral drugs used in human immunodeficiency virus (HIV) care and prevention. Measurement of nucleotide analogs, included in most HIV medication regimens, enables monitoring of short- and long-term adherence and the risk of treatment failure. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay rapidly infers the concentration of intracellular nucleotide analogs based on the inhibition of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Here, we introduce a probabilistic predictive model for RESTRICT and demonstrate selective measurement of multiple nucleotide analogs using DNA templates designed according to the chemical structure of each drug. We measure clinically relevant concentrations of tenofovir diphosphate (TFV-DP), emtricitabine triphosphate (FTC-TP), and azidothymidine triphosphate (AZT-TP) with agreement between experiment and theory. RESTRICT represents a new class of activity-based assays for therapeutic drug monitoring and precision dosing in HIV care and could be extended to other diseases treated with nucleotide analogs.

scholarly journals A Computational Approach for the Prediction of HIV Resistance Based on Amino Acid and Nucleotide Descriptors

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2751 ◽  
Author(s):  
Olga Tarasova ◽  
Nadezhda Biziukova ◽  
Dmitry Filimonov ◽  
Vladimir Poroikov
Keyword(s):  
Machine Learning ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Open Data ◽  
Antiretroviral Drugs ◽  
Amino Acid Sequences ◽  
Machine Learning Techniques ◽  
Structure Property ◽  
Hiv Reverse Transcriptase ◽  
Hiv Resistance

The high variability of the human immunodeficiency virus (HIV) is an important cause of HIV resistance to reverse transcriptase and protease inhibitors. There are many variants of HIV type 1 (HIV-1) that can be used to model sequence-resistance relationships. Machine learning methods are widely and successfully used in new drug discovery. An emerging body of data regarding the interactions of small drug-like molecules with their protein targets provides the possibility of building models on “structure-property” relationships and analyzing the performance of various machine-learning techniques. In our research, we analyze several different types of descriptors in order to predict the resistance of HIV reverse transcriptase and protease to the marketed antiretroviral drugs using the Random Forest approach. First, we represented amino acid sequences as a set of short peptide fragments, which included several amino acid residues. Second, we represented nucleotide sequences as a set of fragments, which included several nucleotides. We compared these two approaches using open data from the Stanford HIV Drug Resistance Database. We have determined the factors that modulate the performance of prediction: in particular, we observed that the prediction performance was more sensitive to certain drugs than a type of the descriptor used.

scholarly journals Monographs on drugs which are frequently analyzed in therapeutic drug monitoring/Arzneimittel-Monographien für Medikamente, die regelmäßig im Rahmen des Therapeutic Drug Monitorings analysiert werden

2012 ◽  
Vol 36 (2) ◽  
Keyword(s):  
Therapeutic Drug Monitoring ◽  
Drug Monitoring ◽  
Clinical Chemistry ◽  
Parent Drug ◽  
Life Time ◽  
Antiretroviral Drugs ◽  
Blood Sampling ◽  
Therapeutic Drug ◽  
Drug Concentrations ◽  
The Stability

AbstractIn addition to the monographs which have been published in the past 7 years by the working group “Drug Monitoring” of the Swiss Society of Clinical Chemistry (SSCC) [1–6], new monographs have been written. The data presented in these monographs provide an overview of the information which is important for the request and interpretation of the results. Therefore, laboratory health professionals and the receivers of the reports are the targeted readers. In this series, antiretroviral drugs are presented for which drug concentrations are regularly determined (protease inhibitors, non-nucleoside reverse transcriptase inhibitors). To date, no clear evidence has been established that therapeutic drug monitoring of these drugs increases the success of the antiretroviral therapy. Nevertheless, many cases have demonstrated that the therapy can be guided with much more confidence and with good success if the drug concentrations are determined, especially if the patient has a combination therapy with many pharmacokinetically interfering compounds. First, information is given about pharmacology and pharmacokinetics of these drugs, such as protein binding, metabolic pathways with specific enzymes involved, elimination half-life time, elimination route(s) of the parent drug, as well as therapeutic and toxic concentrations. Moreover, indications for therapeutic drug monitoring are listed with important preanalytical information (time point of blood sampling and time to steady state since beginning or after change of posology). Furthermore, the stability of the drug and its metabolite(s) after blood sampling are described. For readers with a specific interest, references to important publications are given. The number of monographs will be further enlarged. The updated files are presented on the homepage of the SSCC (www.sscc.ch). We hope that these monographs are helpful for the better handling of therapeutic drug monitoring and we are looking forward to receiving comments from the readers.

scholarly journals Pilot Evaluation of an Enzymatic Assay for Rapid Measurement of Antiretroviral Drug Concentrations

2020 ◽  
Author(s):  
Ayokunle O. Olanrewaju ◽  
Benjamin P. Sullivan ◽  
Ashley R. Bardon ◽  
Tiffany J. Lo ◽  
Tim R. Cressey ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Medical Center ◽  
A Priori ◽  
Reverse Transcriptase Activity ◽  
Enzymatic Assay ◽  
Hiv Reverse Transcriptase ◽  
Drug Concentrations ◽  
Liquid Chromatography Tandem Mass ◽  
Intercalating Dye ◽  
Exposure Prophylaxis

Abstract Objective: Maintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations – a metabolite that indicates long-term PrEP adherence.Setting: The study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle.Methods: We enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader—stronger fluorescence indicated higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using ≥700 fmol/punch TFV-DP as a threshold for adequate adherence (≥4 doses/week).Results: Among 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥700 fmol/punch by LC-MS/MS. RESTRICT fluorescence correlated with LC-MS/MS measurements (r=-0.845, p<0.0001). Median fluorescence was 93.3 (95% CI: 90.9 to 114) for samples <700 fmol/punch and 54.4 (95% CI: 38.0 to 72.0) for samples ≥700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity. Conclusions: This novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

scholarly journals Low Frequency of Drug-Resistant Variants Selected by Long-Acting Rilpivirine in Macaques Infected with Simian Immunodeficiency Virus Containing HIV-1 Reverse Transcriptase

2015 ◽  
Vol 59 (12) ◽  
pp. 7762-7770 ◽  
Author(s):  
Kevin Melody ◽  
Sarah McBeth ◽  
Christopher Kline ◽  
Angela D. M. Kashuba ◽  
John W. Mellors ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Simian Immunodeficiency Virus ◽  
Prolonged Exposure ◽  
Peak Plasma ◽  
Antiretroviral Drugs ◽  
Drug Resistant ◽  
Drug Concentrations ◽  
Immunodeficiency Virus ◽  
Long Acting ◽  
Hiv 1

ABSTRACTPreexposure prophylaxis (PrEP) using antiretroviral drugs is effective in reducing the risk of human immunodeficiency virus type 1 (HIV-1) infection, but adherence to the PrEP regimen is needed. To improve adherence, a long-acting injectable formulation of the nonnucleoside reverse transcriptase (RT) inhibitor rilpivirine (RPV LA) has been developed. However, there are concerns that PrEP may select for drug-resistant mutations during preexisting or breakthrough infections, which could promote the spread of drug resistance and limit options for antiretroviral therapy. To address this concern, we administered RPV LA to macaques infected with simian immunodeficiency virus containing HIV-1 RT (RT-SHIV). Peak plasma RPV levels were equivalent to those reported in human trials and waned over time after dosing. RPV LA resulted in a 2-log decrease in plasma viremia, and the therapeutic effect was maintained for 15 weeks, until plasma drug concentrations dropped below 25 ng/ml. RT mutations E138G and E138Q were detected in single clones from plasma virus in separate animals only at one time point, and no resistance mutations were detected in viral RNA isolated from tissues. Wild-type and E138Q RT-SHIV displayed similar RPV susceptibilitiesin vitro, whereas E138G conferred 2-fold resistance to RPV. Overall, selection of RPV-resistant variants was rare in an RT-SHIV macaque model despite prolonged exposure to slowly decreasing RPV concentrations following injection of RPV LA.

scholarly journals Pilot evaluation of an enzymatic assay for rapid measurement of antiretroviral drug concentrations

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ayokunle O. Olanrewaju ◽  
Benjamin P. Sullivan ◽  
Ashley R. Bardon ◽  
Tiffany J. Lo ◽  
Tim R. Cressey ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Medical Center ◽  
A Priori ◽  
Reverse Transcriptase Activity ◽  
Enzymatic Assay ◽  
Hiv Reverse Transcriptase ◽  
Drug Concentrations ◽  
Liquid Chromatography Tandem Mass ◽  
Intercalating Dye ◽  
Exposure Prophylaxis

Abstract Objective Maintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations—a metabolite that indicates long-term PrEP adherence. Setting The study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle. Methods We enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader—stronger fluorescence indicated higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC–MS/MS) using ≥ 700 fmol/punch TFV-DP as a threshold for adequate adherence (≥ 4 doses/week). Results Among 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥ 700 fmol/punch by LC–MS/MS. RESTRICT fluorescence correlated with LC–MS/MS measurements (r = − 0.845, p < 0.0001). Median fluorescence was 93.3 (95% confidence interval [CI] 90.9 to 114) for samples < 700 fmol/punch and 54.4 (CI 38.0 to 72.0) for samples ≥ 700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity. Conclusions This novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

scholarly journals Pilot Evaluation of an Enzymatic Assay for Rapid Measurement of Antiretroviral Drug Concentrations

2021 ◽  
Author(s):  
Ayokunle O. Olanrewaju ◽  
Benjamin P. Sullivan ◽  
Ashley R. Bardon ◽  
Tiffany J. Lo ◽  
Tim R. Cressey ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Medical Center ◽  
A Priori ◽  
Reverse Transcriptase Activity ◽  
Enzymatic Assay ◽  
Hiv Reverse Transcriptase ◽  
Drug Concentrations ◽  
Liquid Chromatography Tandem Mass ◽  
Intercalating Dye ◽  
Exposure Prophylaxis

Abstract Objective: Maintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations – a metabolite that indicates long-term PrEP adherence.Setting: The study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle.Methods: We enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader—stronger fluorescence indicated higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using ≥700 fmol/punch TFV-DP as a threshold for adequate adherence (≥4 doses/week).Results: Among 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥700 fmol/punch by LC-MS/MS. RESTRICT fluorescence correlated with LC-MS/MS measurements (r=-0.845, p<0.0001). Median fluorescence was 93.3 (95% confidence interval [CI]: 90.9 to 114) for samples <700 fmol/punch and 54.4 (CI: 38.0 to 72.0) for samples ≥700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity. Conclusions: This novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3′-azido-2′,3′-deoxythymidine-resistant HIV isolates

2002 ◽  
Vol 35 (3) ◽  
pp. 155 ◽  
Author(s):  
Xing-Wu Shao ◽  
Sandra Hjalmarsson ◽  
Johan Lennerstrand ◽  
Bo Svennerholm ◽  
Jonas Blomberg ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Chain Termination
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