Human T-lymphocyte Cell Research Articles

Evaluating HIV-1 Infectivity and Virion Maturation across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.

Stain-Free Approach to Determine and Monitor Cell Heath Using Supervised and Unsupervised Image-Based Deep Learning

Cell-based medicinal products (CBMPs) are a growing class of therapeutics that promise new treatments for complex and rare diseases. Given the inherent complexity of the whole human cells comprising CBMPs, there is a need for robust and fast analytical methods for characterization, process monitoring, and quality control (QC) testing during their manufacture. Existing techniques to evaluate and monitor cell quality typically constitute labor-intensive, expensive, and highly specific staining assays. In this work, we combine image-based deep learning with flow imaging microscopy (FIM) to predict cell health metrics using cellular morphology “fingerprints” extracted from images of unstained Jurkat cells (immortalized human T-lymphocyte cells). A supervised (i.e., algorithm trained with human-generated labels for images) fingerprinting algorithm, trained on images of unstained healthy and dead cells, provides a robust stain-free, non-invasive, and non-destructive method for determining cell viability. Results from the stain-free method are in good agreement with traditional stain-based cytometric viability measurements. Additionally, when trained with images of healthy cells, dead cells and cells undergoing chemically induced apoptosis, the supervised fingerprinting algorithm is able to distinguish between the three cell states, and the results are independent of specific treatments or signaling pathways. We then show that an unsupervised variational autoencoder (VAE) algorithm trained on the same images, but without human-generated labels, is able to distinguish between samples of healthy, dead and apoptotic cells along with cellular debris based on learned morphological features and without human input. With this, we demonstrate that VAEs are a powerful exploratory technique that can be used as a process monitoring analytical tool.

Anticancer effects of herbal medicine Emelia-M, Mshikazi and Delosma H against human leukaemia cells.

Leukaemia is one of the three major types of blood cancers that lead to the overproduction of abnormal white blood cells. Emelia M (EMB), Mshikazi and Delosma H are herbal medicines that are being used by traditional healers in KwaZulu-Natal, South Africa to treat leukaemia and other diseases. To gain insight into the safety (non-toxic effect), anti-cancer activity, mechanisms of action and phytochemical profiles of traditional herbal medicines (Emelia M (EMB), Mshikazi and Delosma H) in South Africa. The viability of human peripheral blood mononuclear cells (PBMCs), monocytic (THP-1) and T-lymphocyte (Jurkat) cell lines exposed to varying concentrations of aqueous extracts of the three herbal medicines were assessed using adenosine triphosphate (ATP) assay. All three extracts showed a dose-dependent effect on the viability of PBMCs. Cell viability decreased with increasing concentrations of extracts when compared with the untreated cells at 24 and 48 hours. The inhibitory activities (IC50) of the extract were found in the order of Mshikazi > EMB, > Delosma H. All the extracts induced apoptosis with minimal necrosis. Many bioactive compounds that have been previously reported to have anticancer effects were identified in the extracts. Mshikazi extract significantly inhibited the growth of THP-1 and Jurkat cells and induced cell death through apoptosis than the other two extracts.

A Quenched Size-Expanded Nucleotide Reports Activity of the Leukemia Biomarker Terminal Deoxynucleotidyl Transferase (TdT).

Terminal deoxynucleotidyl Transferase (TdT) is a template-independent DNA polymerase that plays an essential role in the human adaptive immune system and is upregulated in several types of leukemia. It has therefore gained interest as a leukemia biomarker and potential therapeutic target. Herein, we describe a FRET-quenched fluorogenic probe based on a size-expanded deoxyadenosine that reports directly on TdT enzymatic activity. The probe enables real-time detection of primer extension and de novo synthesis activity of TdT and displays selectivity over other polymerase and phosphatase enzymes. Importantly, TdT activity and its response to treatment with a promiscuous polymerase inhibitor could be monitored in human T-lymphocyte cell extract and Jurkat cells using a simple fluorescence assay. Finally, employing the probe in a high-throughput assay resulted in the identification of a non-nucleoside TdT inhibitor.

Data augmentation with improved regularisation and sampling for imbalanced blood cell image classification

Due to progression in cell-cycle or duration of storage, classification of morphological changes in human blood cells is important for correct and effective clinical decisions. Automated classification systems help avoid subjective outcomes and are more efficient. Deep learning and more specifically Convolutional Neural Networks have achieved state-of-the-art performance on various biomedical image classification problems. However, real-world data often suffers from the data imbalance problem, owing to which the trained classifier is biased towards the majority classes and does not perform well on the minority classes. This study presents an imbalanced blood cells classification method that utilises Wasserstein divergence GAN, mixup and novel nonlinear mixup for data augmentation to achieve oversampling of the minority classes. We also present a minority class focussed sampling strategy, which allows effective representation of minority class samples produced by all three data augmentation techniques and contributes to the classification performance. The method was evaluated on two publicly available datasets of immortalised human T-lymphocyte cells and Red Blood Cells. Classification performance evaluated using F1-score shows that our proposed approach outperforms existing methods on the same datasets.

Cytotoxicity and anti-HIV activities of extracts of the twigs of Croton dichogamus\xa0Pax

BackgroundAcquired immunodeficiency syndrome (AIDS) is a clinical syndrome resulting from infection with human immunodeficiency virus (HIV), which causes profound immunosuppression. Anti-HIV drugs that are currently available are chemically synthesized and are frequently limited by side effects, the emergence of drug resistance, affordability, and availability, with over 5 million people in the world lacking access to treatment. As a result, to discover new anti-HIV agents, we investigated the effects of Kenyan C. dichogamus extracts on the laboratory-adapted strain HIV-1IIIB in human T-lymphocytic MT-4 cells.MethodsFour soluble fractions of 1:1 v/v CH2Cl2:MeOH extract of the twigs of C. dichogamus Pax were tested for their replication inhibition activity against the laboratory-adapted strain HIV-1IIIB in the human T-lymphocytic MT-4 cell line. The plant extracts were further evaluated for their cytotoxicity in MT-4 cells using the MTT assay.ResultsThe cytotoxicity CC50 values of the methanol and methylene chloride soluble fractions of C. dichogamus were found to be between 19.58 ± 0.79 and 167 ± 0.8 µg/ml, respectively. The hexane, methylene chloride, and methanol soluble fractions of the 1:1 v/v CH2Cl2:MeOH extract of the twigs of C. dichogamus showed inhibition of the HIV-1IIIB laboratory-adapted strain in a virus-infected cell culture antiviral assay. The methanol soluble fraction of the 1:1 v/v CH2Cl2:MeOH extract of the twigs of C. dichogamus showed significant anti-HIV activity by inhibiting more than 90% of viral-induced cytopathic effects with an IC50 value of 0.06 ± 0.01 µg/ml, giving an SI of 318.5.ConclusionBased on our findings, the methanol soluble fraction of the 1:1 v/v CH2Cl2:MeOH extract of the twigs of C. dichogamus has shown potential efficacy in inhibiting viral replication and could be considered a promising candidate for further studies.

Design, Synthesis, Spectroscopic Characterisation and In Vitro Cytostatic Evaluation of Novel Bis(coumarin-1,2,3-triazolyl)benzenes and Hybrid Coumarin-1,2,3-triazolyl-aryl Derivatives.

In this work, a series of novel 1,2,3-triazolyl-coumarin hybrid systems were designed as potential antitumour agents. The structural modification of the coumarin ring was carried out by Cu(I)-catalysed Huisgen 1,3-dipolar cycloaddition of 7-azido-4-methylcoumarin and terminal aromatic alkynes to obtain 1,4-disubstituted 1,2,3-triazolyl-coumarin conjugates 2a–g, bis(1,2,3-triazolyl-coumarin)benzenes 2h–i and coumarin-1,2,3-triazolyl-benzazole hybrids 4a–b. The newly synthesised hybrid molecules were investigated for in vitro antitumour activity against five human cancer cell lines, colon carcinoma HCT116, breast carcinoma MCF-7, lung carcinoma H 460, human T-lymphocyte cells CEM, cervix carcinoma cells HeLa, as well as human dermal microvascular endothelial cells (HMEC-1). Most of these compounds showed moderate to pronounced cytotoxic activity, especially towards MCF-7 cell lines with IC50 = 0.3–32 μM. In addition, compounds 2a–i and 4a–b were studied by UV-Vis absorption and fluorescence spectroscopy and their basic photophysical parameters were determined.

In Vitro Cytotoxicity and Anti-HIV Activity of Crude Extracts of Croton macrostachyus, Croton megalocarpus and Croton dichogamus.

IntroductionHuman immunodeficiency virus (HIV) affects the body’s defense mechanisms and leads to a number of opportunistic infections which later cause fatality as a result of an acquired immunodeficiency syndrome (AIDS). More than half a million individuals have lost their life in 2020 due to this disease. Antiretroviral drugs have played a great role in improving the quality of life of HIV infected individuals. The side effects of these drugs coupled with resistance of the virus to the various regimens, necessitates the search for potentially new and effective antiretroviral medication. The objective of this study is to evaluate anti-HIV activity of crude extracts of three Croton plants.MethodsAs part of our effort in screening anti-HIV medications, we evaluated the cytotoxicity and anti-HIV activity of three Croton species used as herbal medicine in Africa. Crude extracts of Croton macrostachyus, Croton megalocarpus and Croton dichogamus were tested for their replication inhibition activity against laboratory adapted strains HIV-1IIIB in Human T-lymphocytic MT-4 cell line.ResultsBased on our findings, the crude aerial part extract of C. dichogamus displayed the highest anti-HIV activity by inhibiting 73.74% of viral induced cytopathic effect (CPE) at IC50 value of 0.001 + 0.00 μg/mL giving a selectivity index (SI) of 3116.0. In addition, the crude leaf extract of C. megalocarpus showed higher anti-HIV activity by inhibiting 74.65% of CPE at IC50 value of 0.05 + 0.03 μg/mL giving an SI of 571.3.ConclusionOut of five extracts from three Croton species screened for anti-HIV activity using human T-lymphocytic MT-4 cells, the leaf extract of Croton megalocarpus and aerial part extract of Croton dichogamus could be considered as promising extracts as they display high antiviral activity with low toxicity and high selectivity index values. To investigate the active constituents responsible for the anti-HIV activity, chemical identification of the active constituents is now in progress in our laboratory. Since there is no previously reported anti-HIV activity for these plants, there is a great need to isolate the compounds responsible for the noted activity.

Synthesis, Molecular Docking and Preliminary Antileukemic Activity of 4-Methoxybenzyl Derivatives Bearing Imidazo[2,1-b][1,3,4]thiadiazole.

In this study, we synthesized 22 compounds in a series with various substitution on imidazo[2,1-b][1,3,4]thiadiazole. The potential cytotoxic activity of these compounds investigated in leukemia cell lines by Differential Nuclear Staining (DNS). Our results identified two compounds, 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate and 6-(4-chlorophenyl)-2-(4-methoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde, exhibited the most cytotoxic effect against murine leukemia cells (L1210), human T-lymphocyte cells (CEM) and human cervix carcinoma cells (HeLa) with IC50 values ranging between 0.79 and 1.6 μM. The results indicate that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate is inducing phosphatidylserine externalization and caspase-3 activation which are both a hallmark of apoptosis. Docking studies showed that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate binds within the active sites of transforming growth factor beta (TGF-β) type I receptor kinase domain by strong hydrogen binding and hydrophobic interactions.

Biological Properties of New Chiral 2-Methyl-5,6,7,8-tetrahydroquinolin-8-amine-based Compounds.

The synthesis of a small library of 8-substituted 2-methyl-5,6,7,8-tetrahydroquinoline derivatives is presented. All the compounds were tested for their antiproliferative activity in non-cancer human dermal microvascular endothelial cells (HMEC-1) and cancer cells: human T-lymphocyte cells (CEM), human cervix carcinoma cells (HeLa), human dermal microvascular endothelial cells (HMEC-1), colorectal adenocarcinoma (HT-29), ovarian carcinoma (A2780), and biphasic mesothelioma (MSTO-211H). Compounds 3a, 5a, and 2b, showing significant IC50 values against the whole panel of the selected cells, were further synthesized and tested as pure enantiomers in order to shed light on how their stereochemistry might impact on the related biological effect. The most active compound (R)-5a was able to affect cell cycle phases and to induce mitochondrial membrane depolarization and cellular ROS production in A2780 cells.

Effect of Harvest Age on Total Phenolic, Total Anthocyanin Content, Bioactive Antioxidant Capacity and Antiproliferation of Black and White Glutinous Rice Sprouts

Black (cv. BGR) and white (cv. RD6) glutinous rice sprouts from fertilizer- and pesticide-free farm in Khon Kaen province, Thailand were investigated for antioxidation and antiproliferative activity. Three different ages of rice sprouts were collected and prepared as the extract. BGR exerted higher antioxidant capacity than RD6 based on total phenolic (TPC) and total anthocyanin contents (TAC), DPPH, and FRAP assays. BGR at 10–15 days contained the highest TPC (29.72 ± 1.42 mg gallic acid equivalent/g extract) and reducing power (2.22 ± 0.014 mmole FeSO4/g extract). BGR at 20–25 days contained the highest TAC (0.86 ± 0.096 equivalence of cyanidin-3-glucoside/g extract) and DPPH radical scavenging activity (IC50 = 231.09 ± 12.99 μg/mL). Antiproliferative activity of the extracts was evaluated in the human T-lymphocyte (Jurkat), hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT116), melanoma (SK-MEL-2) and noncancerous cells (Vero) by neutral red assay. BGR showed the most selective antiproliferation against Jurkat cells, by inducing apoptosis, and caspase 3/7 activity. BGR at 200 μg/mL from all ages significantly decreased ROS using DCFH-DA and increased endogenous glutathione levels in Jurkat cells compared to the control (p &lt; 0.05). The higher antiproliferation of BGR than RD6 was via its antioxidation capacity and attributed to its higher phenolic and anthocyanin contents. BGR sprout is a potential source of biologically active substances good for wellness and health benefits.

Discovery of phenylselenoether-hydantoin hybrids as ABCB1 efflux pump modulating agents with cytotoxic and antiproliferative actions in resistant T-lymphoma

Multidrug resistance (MDR) in cancer cells is a crucial aspect to consider for a successful cancer therapy. P-gp/ABCB1, a member of ABC transporters, is involved in the main tumour MDR mechanism, responsible for the efflux of drugs and cytotoxic substances. Herein, we describe a discovery of potent selenium-containing ABCB1 MDR efflux pump modulators with promising anticancer activity. On three groups of selenoethers comprehensive studies in terms of design, synthesis, and biological assays, including an insight into cellular mechanisms of anticancer action as well as an ADMET-screening in vitro were performed, followed by in-depth SAR analysis. Among the investigated new phenylselenoether hybrids, four compounds showed significant cytotoxic and anti-proliferative effects, in particular, in resistant cancer cells. Hydantoin derivatives (5–7) were significantly more effective than the reference inhibitor verapamil (up to 2.6-fold at a 10-fold lower concentration) modulating ABCB1-efflux pump, also possessing a good drug-drug interaction profile. The best compound (6) was further evaluated in human JURKAT T-lymphocytic cancer cells for its impact on cell proliferation rate. Mechanistically, the expression of cyclin D1, an enhancer of the cell cycle, decreases, while p53, an inhibitor of cell proliferation, was up-regulated upon the treatment with compound 6 alone or in combination with the chemotherapeutic agent doxorubicin. In summary, a new chemical space of highly active selenium-containing anticancer agents has been discovered, with a new lead compound 6 that warrants more in-depth biological evaluation and further pharmacomodulation.

Mechanical Low-Pass Filtering of Cells for Detection of Circulating Tumor Cells in Whole Blood.

The detection of circulating tumor cells (CTCs) from liquid biopsies using microfluidic devices is attracting a considerable amount of attention as a new, less-invasive cancer diagnostic and prognostic method. One of the drawbacks of the existing antibody-based detection systems is the false negatives for epithelial cell adhesion molecule detection of CTCs. Here we report a mechanical low-pass filtering technique based on a microfluidic constriction and electrical current sensing system for the novel CTC detection in whole blood without any specific antigen-antibody interaction or biochemical modification of the cell surface. The mechanical response of model cells of CTCs, such as HeLa, A549, and MDA-MB-231 cells, clearly demonstrated different behaviors from that of Jurkat cells, a human T-lymphocyte cell line, when they passed through the 6-μm wide constriction channel. A 6-μm wide constriction channel was determined as the optimum size to identify CTCs in whole blood with an accuracy greater than 95% in tens of milliseconds. The mechanical filtering of cells at a single cell level was achieved from whole blood without any pretreatment (e.g., dilution of lysing) and prelabeling (e.g., fluorophores or antibodies).

Label-free purification of viable human T-lymphocyte cells from a mixture of viable and non-viable cells after transfection by electroporation

Recent developments in chimeric antigen receptor (CAR) T-cell therapy for cancer have shown promising results. CAR T-cells are engineered mainly by AC or DC electroporation to express CAR molecules on their surfaces. Electroporation has detrimental effects on the majority of T-cells and causes cell death via apoptosis or necrosis. These non-viable cells have potentially harmful effects on other healthy T-cells. Because there are no unique biomarkers that pinpoint the apoptosis and necrosis of T-cells, currently available cell separation methods are incapable of purifying healthy T-cells after electroporation. To address this critical issue, we have used dielectrophoretic cell isolation in a simple microfluidics chip. Our results indicate that it is possible to purify cell samples after electroporation and achieve ~100% purity and >90% target cell recovery. Therefore, this work presents a viable solution to a critical need in CAR T-cell manufacturing.

Imidazolium-Sulfobetaine Based on Betulin with Proliferative Activity for HEK293 and T-Lymphocyte Cells

A betulin derivative with an (imidazolium)butane-1-sulfonate fragment was synthesized and promoted proliferation in vitro of HEK293 and human T-lymphocyte cells, suggesting that this compound had immunostimulant properties.

Synergistic therapeutic effect of diethylstilbestrol and CX-4945 in human acute T-lymphocytic leukemia cells

Human acute T-lymphocytic leukemia (T-ALL) is one of the most commonly diagnosed hematological disorders, and is characterized by poor prognosis and survival rate. Despite the development of new therapeutic approaches, leukemia treatment options remain limited. In this study, we investigated the immunosuppressive and anti-proliferative effects of the synthetic estrogen diethylstilbestrol (DES), both alone and combined with the casein kinase 2 (CK2) inhibitor CX-4945. Our results indicated that DES induced caspase-dependent apoptosis in a human T-ALL cell line (Jurkat cells), while exerting no significant cytotoxicity in normal peripheral blood mononuclear cells (PBMCs). Phytohaemagglutinin and phorbol 12-myristate 13-acetate induced interleukin (IL)-2 production and activation of NF-κB signaling pathways, which were both inhibited by DES. Moreover, DES exerted synergistic effects with CX-4945 on proliferation and IL-2 production in Jurkat cells. Our results demonstrated that DES exerts anti-proliferative and immunosuppressive effects through inhibition of CK2 and the NF-κB signaling pathway in human T-ALL Jurkat cells.

QSAR studies of indoyl aryl sulfides and sulfones as reverse transcriptase inhibitors

The inhibitory HIV reverse transcriptase activity of 172 non-nucleoside indoyl aryl sulfones and sulfides is studied with a QSAR analysis, in order to identify the molecular characteristics influencing the interaction with the reverse transcriptase enzyme. This work increases the available QSAR studies of indoyl aryl sulfones and sulfides using the reported experimental EC50 values against HIV-1 wild type (IIIB) in human T-lymphocyte (CEM) cells. Different approaches are proposed, involving 0D, 1D and 2D molecular descriptors from PaDEL freeware, and also based on flexible descriptors from CORAL freeware. Three models are finally presented, which correlate the inhibitory HIV reverse transcriptase activity with good accuracy. It is demonstrated that the established models are predictive in the validation process. The novelty of the present work relies on the development of structure-inhibitory HIV activity relationships, through a computational technique that does not require the knowledge of the molecular conformation during the structural representation. The obtained results would contribute to guide the design of more effective compounds for HIV treatment.

Design, synthesis, structural characterization and in vitro cytotoxic activity of mononuclear Ru(II)complexes

The synthesis and characterization of ruthenium complexes (Ru-1–Ru-6) of the type [Ru(R)2(K)]2+ (where R = 1,10-phenanthroline/2,2′-bipyridyl and K = acetyl coumarin-inh, pyrazole-tch, acetyl coumarin-tsz, are described. These ligands form bidentate octahedral ruthenium complexes. The in vitro cytotoxic activities of the complexes measurement against the human cancer T-lymphocyte cell lines. In vitro evaluation of these title complexes revealed cytotoxicity from 0.34 to 1.4 µg/mL against CEM, 0.28 to 1.8 µg/mL against L1210, 0.44 to 2.5 µg/mL against Molt4/C8, 0.98 to 1.6 µg/mL against HL60, and 0.66 to 1.4 µg/mL against BEL7402. Ruthenium complexes Ru-5 & Ru-6 showed that quite significant anticancer activities over standard drugs.

Synthesis of Novel Chiral Sulfonamide‐Bearing 1,2,4‐Triazole‐3‐thione Analogs Derived fromD‐andL‐Phenylalanine Esters as Potential Anti‐Influenza Agents

Novel enantiopure 1,2,4-trizole-3-thiones containing a benzensulfonamide moiety were synthesized via multistep reaction sequence starting with D-phenylalanine methyl ester and L-phenylalanine ethyl ester as a source of chirality. The chemical structures of all compounds were characterized by elemental analysis, UV, IR, (1) H NMR, (13) C NMR, 2D NMR (HETCOR), and mass spectral data. All compounds were tested in vitro antiviral activity against a broad variety of DNA and RNA viruses and in vitro cytostatic activity against murine leukemia (L1210), human T-lymphocyte (CEM) and human cervix carcinoma (HeLa) cell lines. Although enantiopure 1,2,4-triazole-3-thione analogs in (R) configuration emerged as promising anti-influenza A H1N1 subtype in Madin Darby canine kidney cell cultures (MDCK), their enantiomers exhibited no activity. Especially compounds , , , , and (EC50 : 6.5, 6.1, 2.4, 1.6, 1.7μM, respectively) had excellent activity against influenza A H1N1 subtype compared to the reference drug ribavirin (EC50 : 8.0μM). Several compounds have been found to inhibit proliferation of L1210, CEM and HeLa cell cultures with IC50 in the 12-53μM range. Compound and in (R) configuration were the most active compounds (IC50 : 12-22μM for and IC50 : 19-23μM for ). Chirality 28:495-513, 2016. © 2016 Wiley Periodicals, Inc.

Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues.

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.