In this study, we isolated and structurally characterized a diastereoisomer (1- R) of the betulonic acid-zidovudine (AZT) conjugates 1- S , a previously reported anticancer lead. The absolute configuration at the C-2 position was determined via 1D/2D nuclear magnetic resonance (NMR) and optical rotatory dispersion (ORD). Both diastereomers exhibited potent anticancer activity against a panel of human cancer cell lines, with particular efficacy against SMMC-7721 (hepatoma) and A549 (lung carcinoma) cells (IC50 = 3.38-4.09 μM). Mechanistic investigations revealed that 1- R induces cell death primarily through a caspase-dependent apoptotic pathway. Notably, the necroptosis inhibitor necrostatin-1 (Nec-1, an inhibitor of necroptosis) provided substantial protection, suggesting that necroptosis may play a more prominent role in 1- R compared to that of 1- S , although this requires direct comparative validation in future studies. The autophagy inhibitor 3-methyladenine (3-MA, an autophagy inhibitor) partially attenuated cell death, indicating a modulatory role for autophagy. These findings highlight 1- R as a promising anticancer agent. Despite its initial isolation as a low-yield byproduct (3.2%), the comparable activity of 1- R to 1- S , coupled with the conformational plasticity of the C-2 linker, simplifies synthetic requirements and supports its further optimization.

Adult T-cell leukemia (ATL) is a rare but aggressive CD4[+]CD25[+] leukemia triggered by HTLV-1 infection. We quantified ex vivo levels of CD4[+] and CD8[+] (sub)populations and their antiproliferative, pro-apoptotic, antiviral and immunomodulatory interplay in short-term culture of primary cells from ATL patients, in a long-term prospective study (819 person-years of follow-up). We integrated clinical, cellular and molecular data into a data mining approach combining linear (consensus HIerarchical Tree clustering) and non-linear (BAYesian network) models. This HIT-BAY approach revealed an association between CD8[+]CD25[+] cells and survival, independent of CD4[+]CD25[+] cells. Moreover, CD8[+]CD25[+] levels at diagnosis significantly predicted 5-year survival in ATL patients (p = 0.037), which was confirmed in multivariable Cox regression models correcting for clinical forms. We provide in vivo support for our ex vivo model in a unique patient on AZT monotherapy, for whom adding IFN-α resulted in a rapid (<2 weeks) and pronounced (>70%) decline in leukemic/non-leukemic cell ratio, accompanied by a ten-fold increase CD8[+]CD25[+] levels, and followed by long-term survival. Finally, transcriptomic analysis of a CD8[+]CD25[+] gene module and replication in an independent ATL cohort revealed shared cytotoxic activity of both CD8[+]CD25[+] and Tax-specific CD8[+] cells as an underlying mechanism for prolonged survival. In conclusion, our integrated data mining approach allowed us to integrate ex vivo clinical and immunological data, revealing a central role for CD8[+]CD25[+] cells in ATL survival. HIT-BAY might serve as a prototype to model immune response and facilitate biomarker and therapeutic target discovery in real-world cohorts, including rare malignancies.

Near-infrared hyperspectral imaging (NIR-HSI) was evaluated as a rapid, non-destructive screening tool for the detection of harmful toxin-producing microalgae directly in natural seawater samples. One year of weekly to bi-weekly collected samples (May 2020-June 2021) from two stations in Ria de Aveiro lagoon (Portugal) were analysed, covering strong seasonal and inter-annual variability in phytoplankton assemblages. Hyperspectral images of filtered seawater samples were recorded in the 900-1700 nm range and processed by multivariate image analysis. Cell counts and taxonomic identification were obtained using the reference Utermöhl inverted microscopy method for species producers of AST, PST, DST, YTX and AZT toxins. PLS-DA models built on mean image spectra enabled to distinguish between samples above and below the detection limits of the reference method for all five toxin-producers. Cross-validated accuracies reached 0.98 for AST and PST producers and 0.87-0.90 for DST, AZT and YTX producers. NIR-HSI demonstrated superior performance compared with FT-MIR spectroscopy for AST, PST and DST groups. Furthermore, NIR-HSI allowed species-level discrimination within Dinophysis, with accuracies up to 0.98 for D. ovum. The results demonstrate that NIR-HSI can detect toxic microalgae at ecologically relevant abundances and has strong potential as a screening technology for HAB monitoring programmes.

The Chlamydiota phylum consists of obligate intracellular bacteria, including well-known pathogens and emerging environmental species, with diverse host ranges and metabolic capabilities. Among these bacteria, the gene which encodes nucleoside diphosphate kinase, ndk is present in variable numbers. While most chlamydial species carry a single paralog of ndk, some species have two paralogs. In Waddlia chondrophila, the two Ndk proteins encoded by ndk paralogs retain conserved kinase motifs but differ in subcellular localization, suggesting divergent functional roles. According to localization studies performed in heterologous expression systems, WcNdk1 is confined to the bacteria-containing vacuole and probably supports nucleotide metabolism, while WcNdk2 localizes to the host nucleus, perinuclear space, and Golgi apparatus, suggesting involvement in host interaction. Azidothymidine (AZT), a known Ndk inhibitor, impaired W. chondrophila growth, potentially through inhibition of WcNdk2. However, the lack of genetic tools currently limits definitive functional conclusions. Our data suggests potential functions for Ndks in W. chondrophila, providing a foundation for future studies on Ndk-mediated interactions between this pathogen and its host, which might possibly be translated into a new therapeutic approach.

Background/Objectives: Despite the presumption of bioequivalence for BCS Class I drugs due to their high solubility and permeability, recent evidence indicates that those with rapid systemic elimination exhibit heightened vulnerability to Cmax non-equivalence, primarily attributable to intrasubject variability in gastrointestinal transit and absorption kinetics. It is well known that gastric emptying is a significant physiological-dependent factor. But, does the formulation affect gastric emptying? Methods: Using zidovudine as a model drug, formulations containing sodium carboxymethyl starch (CMS-Na), pregelatinized starch, hydroxypropyl methylcellulose (HPMC), and lactose were investigated for their effects on gastric emptying kinetics, and the impact of excipient-mediated gastric emptying prolongation on pharmacokinetic parameters was also evaluated. Results: Relative to AZT alone (Cmax = 13,350 ng/mL; gastric %ID = 11.3%), co-administration with CMS-Na, pregelatinized starch, or HPMC significantly prolonged gastric retention (%ID: 23.4%, 30.5%, and 40.8% at 22.5 min) and reduced Cmax in rats by 47.8%, 34.4%, and 35.1%, respectively, with no effect on intestinal permeability. Viscosity positively correlated with gastric emptying delay. Conclusions: Our rat findings provide new possible mechanistic evidence that certain viscosity-modifying excipients can delay gastric emptying and reduce Cmax of zidovudine, a rapidly eliminated BCS Class I drug, with potential implications for biowaiver risk assessment. Gastric emptying is not only a physiological-dependent variation but also, in cases where common excipients may significantly delay gastric emptying, a formulation-dependent rate-limiting step. For such drugs, excipient-induced gastric emptying delay poses an underappreciated risk to the biowaiver approach, necessitating more prudent regulatory assessment that encompasses the dynamic interplay among sequential rate processes governing drug disposition.

Colorectal cancer is the third most commonly diagnosed cancer worldwide and the second leading cause of cancer-related deaths, while its resistance to treatment continues to represent a major therapeutic challenge. In the present study, a series of phenothiazine derivatives, including hybrids containing a 1,2,3-triazole linker and the zidovudine (AZT) fragment, were synthesized and evaluated for their anticancer activity against colorectal cancer cell lines HCT116 and HT-29 as well as non-cancerous BEAS-2B cells. Cytotoxic activity was determined using the Alamar Blue assay, while the mechanisms of action were investigated by flow cytometric analysis of apoptosis, cell cycle progression, and reactive oxygen species (ROS) generation. Additionally, changes in the expression of genes associated with apoptosis, oxidative stress, and DNA damage response were analyzed by RT-qPCR. The obtained results demonstrated that AZT-containing derivatives exhibited stronger anticancer activity than non-conjugated phenothiazine analogs. Compounds A9-A12 induced pronounced apoptosis and significant disturbances in cell cycle progression, particularly in HCT116 cells. Among the analyzed derivatives, compound A9 displayed the most favorable overall biological profile, combining strong proapoptotic and cytotoxic activity with relatively high selectivity toward cancer cells and moderate effects on non-cancerous cells. The results indicate that molecular hybridization of phenothiazine derivatives with the AZT scaffold represents a promising strategy for the development of novel anticancer agents targeting colorectal cancer.

Adult T cell leukemia/lymphoma (ATLL), caused by human T cell leukemia virus type 1, is an aggressive malignancy with limited treatment options. Although the combination therapy of azidothymidine (AZT) and interferon-α (IFN-α) has demonstrated clinical efficacy in ATLL, its underlying mechanism remains unclear. In this study, through comprehensive analyses, we reveal that the combination therapy can enhance CD8+ T cell function both in vitro and in vivo: it promotes cytotoxic T lymphocyte (CTL)-mediated killing of ATLL cell lines, and exhibits antitumor activities in immunocompetent but not immunodeficient mouse xenograft models (MC38 and EL4 were used due to the lack of immunocompetent mouse model for ATLL). Granzyme B, which is downregulated in ATLL patients, was found to be key to the effectiveness of AZT/IFN-α therapy. In addition, AZT promotes IFN-α-mediated antigen processing and presentation, and it further transcriptionally upregulates the expression of the ATLL antigen HBZ. Collectively, AZT could potentiate the immunomodulatory function of IFN-α in ATLL likely via enhancing tumor antigen expression and processing and boosting CTL activities. This study suggests a key role of immunostimulation in AZT/IFN-α-treated ATLL and may provide mechanistic insights into the development of novel therapies.

The NS3 protein of dengue virus (DENV) is highly conserved and plays a vital role in replication, and thus it is considered as a potential antiviral target. The current study was carried out to observe interactions between the NS3 protein of DENV-4 and 3'-azido-3'-deoxythymidine (AZT-TP) using biophysical studies and ATPase inhibition assays, and further complemented it with in silico approaches. Fluorescence and time-resolved studies showed significant static quenching of NS3 fluorophores. Surface plasmon resonance (SPR) studies showed strong binding affinity with a significant pharmacodynamics rate, i.e., higher kinetic rate of association (k a) and lower dissociation rate constant (Kd). Isothermal titration calorimetry (ITC) measurements revealed strong and spontaneous binding between NS3 and AZT-TP with feasible thermodynamic parameters. The ATPase inhibition assay showed a decrease in NS3 ATPase activities in a dose-dependent manner, indicating the inhibitory potential of AZT-TP. Molecular docking showed significant binding (-8.0 kcal/mol) with the conserved residues involved in NS3 helicase/ATPase activities. Molecular dynamics (MD) simulation studies showed no major conformational changes in the NS3 and AZT-TP complex, and it was well-anchored at its binding position with nearly a linear trajectory up to 200 ns. Total Gibbs free energy and contribution of different interactions calculated using MMPB-(GB)-SA complemented the biophysical and docking results. The study suggests that there might be a direct binding between AZT-TP and residues involved in the ATPase activities and thus provides promising data to explore the future prospects of AZT-TP as an NS3 inhibitor for antiviral development.

Faithful DNA replication in Escherichia coli requires the DNA polymerase III holoenzyme (DNA pol III HE) and its clamp loader complex, which couples processive DNA synthesis with β-clamp loading. The clamp loader accessory subunits χ and ψ link single-stranded DNA-binding protein (SSB) to the replisome, stabilizing replication on SSB-coated templates through interactions with the SSB C-terminal tail. Chi has also been implicated in tolerance to the chain-terminating nucleoside analog azidothymidine (AZT), though whether this function depends on χ within DNA pol III HE or on its independent interaction with the YoaA helicase remains unclear. To address this, we engineered ψ-χ fusion proteins with flexible glycine-serine linkers to tether the two subunits while preserving folding and activity. Both fusions were biochemically competent, supporting ATP hydrolysis and clamp loading on SSB-coated DNA. In vivo, however, neither the ψ-GS12-χ fusion nor expression of a ψχ operon restored AZT tolerance in ΔholC cells, whereas expression of χ alone was sufficient. Fusion expression impaired growth in both wild-type (WT) and ΔholC backgrounds, a phenotype alleviated by disrupting χ-SSB binding. These findings support a model in which χ must dynamically engage SSB and YoaA outside of the clamp loader to promote AZT tolerance, highlighting the importance of regulated χ-SSB interactions in genome maintenance.

The widespread use of pharmaceuticals has led to their frequent detection in aquatic environments, raising concerns about adverse effects on non-target organisms. This study examined behavioral and endocrine responses to acute repetitive exposure (4 days, 15 min day-1) to environmentally relevant concentrations of azithromycin (AZT, 12.5 μg mL-1), nimesulide (NIME, 0.4 μg mL-1), and their combination in zebrafish (Danio rerio). Behavior was assessed using the Novel Tank Test, Social Preference Test, and Light/Dark Test. Whole-body cortisol, acetylcholinesterase (AChE) and catalase (CAT) activities, lipid peroxidation (TBARS), and non-protein thiols (NTP) were measured as stress biomarkers. NIME significantly reduced locomotion (∼30%) and time spent in upper zones (p < 0.05), indicating hypolocomotion and anxiety-like behavior. AZT elevated cortisol (p < 0.05) but caused minimal behavioral changes. Co-exposure intensified behavioral impairments and further increased cortisol (p < 0.01), suggesting synergistic effects. NIME increased TBARS, indicating oxidative damage. AChE activity was inhibited in the brain by NIME, while AZT and the combination increased systemic AChE. CAT activity rose in the combination group, possibly reflecting a compensatory response to reactive oxygen species. These findings demonstrate that short-term, repeated exposure to mixtures of commonly detected pharmaceuticals, even at environmentally relevant concentrations, can disrupt behavioral, biochemical, and endocrine homeostasis in aquatic organisms. This highlights the ecological risks posed by multi-compound contamination in aquatic environments.